Nucleic acid based data storage

ABSTRACT

Provided herein are compositions, devices, systems and methods for the generation and use of biomolecule-based information for storage. Additionally, devices described herein for de novo synthesis of nucleic acids encoding information related to the original source information may be rigid or flexible material. Further described herein are highly efficient methods for long term data storage with 100% accuracy in the retention of information. Also provided herein are methods and systems for efficient transfer of preselected polynucleotides from a storage structure for reading stored information.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.16/927,826, filed Jul. 13, 2020, which is a continuation of U.S. patentapplication Ser. No. 16/415,960, filed May 17, 2017, now U.S. Pat. No.10,754,994, issued on Aug. 25, 2020, which is a continuation of U.S.patent application Ser. No. 15/709,274 filed Sep. 19, 2017, now U.S.Pat. No. 10,417,457, issued on Sep. 17, 2019, which claims the benefitof U.S. Provisional Application No. 62/517,671 filed Jun. 9, 2017; U.S.Provisional Application No. 62/446,178 filed Jan. 13, 2017; and U.S.Provisional Application No. 62/397,855 filed Sep. 21, 2016, each ofwhich are incorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 18, 2017, isnamed 44854-728_303_SL.txt and is 1.612 bytes in size.

BACKGROUND

Biomolecule based information storage systems, e.g., DNA-based, have alarge storage capacity and stability over time. However, there is a needfor scalable, automated, highly accurate and highly efficient systemsfor generating biomolecules for information storage.

BRIEF SUMMARY

Provided herein are methods for storing and accessing information, themethod comprising: (a) converting at least one item of information in aform of at least one digital sequence to at least one nucleic acidsequence; (b) providing a structure comprising a surface; (c)synthesizing a plurality of polynucleotides having predeterminedsequences collectively encoding for the at least one nucleic acidsequence, wherein each polynucleotide extends from the surface; (d)storing the plurality of polynucleotides; and (e) selectivelytransferring the plurality of polynucleotides to a receiving unit,wherein selectively transferring comprises application of a force,wherein the force is laminar pressure, capillary pressure, slip flowpressure, magnetic force, electrostatic force, peristaltic force, soundwaves, vibrational force, centripetal force, centrifugal force, or anycombination thereof, and wherein the plurality of polynucleotidescollectively encodes for a single nucleic acid sequence of the at leastone nucleic acid sequence. Further provided herein are methods, whereinthe application of force comprises a conducting member, and an appliedvoltage potential between the structure and the conducting member.Further provided herein are methods, wherein the application of forcecomprises contacting the surface of the structure with a rigid orflexible slip. Further provided herein are methods, wherein theapplication of force comprises a pressure release or pressure nozzle.Further provided herein are methods further comprising using thepressure nozzle during step (c). Further provided herein are methodsfurther comprising flooding the polynucleotides through the pressurenozzle. Further provided herein are methods further comprisingdepositing nucleotides through the pressure nozzle. Further providedherein are methods further comprising: sequencing the plurality ofpolynucleotides; and assembling the at least one digital sequence.Further provided herein are methods, wherein the at least one digitalsequence assembled is 100% accurate compared to an initial at least onedigital sequence.

Provided herein are methods for storing information, the methodcomprising: (a) converting at least one item of information in a form ofat least one digital sequence to at least one nucleic acid sequence; (b)synthesizing a plurality of polynucleotides having predeterminedsequences collectively encoding for the at least one nucleic acidsequence, wherein each polynucleotide comprises: (i) a plurality ofcoding regions, wherein each coding region is identical; and (ii) atleast one non-coding region, wherein the at least one non-coding regioncomprises a cleavage region; and (c) storing the plurality ofpolynucleotides. Further provided herein are methods, wherein thecleavage region comprises a restriction enzyme recognition site. Furtherprovided herein are methods, wherein the cleavage region comprises alight sensitive nucleobase. Further provided herein are methods furthercomprising application of a restriction enzyme, electromagneticradiation, or a gaseous reagent to cleave at the cleavage region,thereby removing at least one of the plurality of coding regions.Further provided herein are methods, wherein each coding regioncomprises 25 to 500 bases in length. Further provided herein aremethods, wherein each coding region comprises 100 to 2000 bases inlength. Further provided herein are methods, wherein each non-codingregion comprises 1 to 100 bases in length. Further provided herein aremethods, wherein each non-coding region comprises at most 200 bases.Further provided herein are methods, wherein the plurality ofpolynucleotides comprises at least 100,000 polynucleotides. Furtherprovided herein are methods, wherein the plurality of polynucleotidescomprises at least 10 billion polynucleotides. Further provided hereinare methods, wherein greater than 90% of the polynucleotides encode fora sequence that does not differ from the predetermined sequence. Furtherprovided herein are methods, wherein the at least one item ofinformation is text information, audio information or visualinformation. Further provided herein are methods, wherein a firstnon-coding region within each polynucleotide has a different sequencethan a second non-coding region within each polynucleotide. Furtherprovided herein are methods, wherein each non-coding region within eachpolynucleotide has a different sequence. Further provided herein aremethods, wherein a first cleavage region within each polynucleotide hasa different sequence than a second cleavage region within eachpolynucleotide. Further provided herein are methods, wherein eachcleavage region within each polynucleotide has a different sequence.Further provided herein are methods, wherein a number of cleavageregions within each polynucleotide is at least 1, 2, 3, 4, or S. Furtherprovided herein are methods, wherein a sequence for the number ofcleavage regions is different. Further provided herein are methods,wherein each polynucleotide comprises a tether region.

Provided herein are methods for encrypting information, the methodcomprising: (a) converting at least one item of information in a form ofat least one digital sequence to at least one nucleic acid sequence; (b)associating each of the at least one nucleic acid sequence with one of aplurality of non-identical markings; (c) providing a structure having asurface, wherein the surface comprises the plurality of non-identicalmarkings; (d) synthesizing a plurality of polynucleotides havingpredetermined sequences collectively encoding for the at least onenucleic acid sequence, wherein the plurality of polynucleotidescomprises at least 100,000 polynucleotides, and wherein eachpolynucleotide extends from the surface in a discrete region demarcatedby one of the non-identical markings; and (e) storing the plurality ofpolynucleotides. Further provided herein are methods, wherein theplurality of polynucleotides comprises at least 1,000,000polynucleotides. Further provided herein are methods, wherein greaterthan 90% of the polynucleotides encode for a sequence that does notdiffer from the predetermined sequence. Further provided herein aremethods, wherein the at least one item of information is textinformation, audio information or visual information. Further providedherein are methods, wherein a subset of the polynucleotides discretelydemarcated by one of the non-identical markings comprise a samesequence. Further provided herein are methods further comprisingselecting a subset of polynucleotides discretely demarcated by one ofthe non-identical markings, releasing the subset of polynucleotides,sequencing the plurality of polynucleotides, decrypting the plurality ofpolynucleotides, and assembling the at least one digital sequence.Further provided herein are methods further comprising selecting asubset of polynucleotides discretely demarcated by one of thenon-identical markings, amplifying the subset of polynucleotides,sequencing the subset of polynucleotides, decrypting the plurality ofpolynucleotides, and assembling the at least one digital sequence.Further provided herein are methods, wherein the at least one digitalsequence assembled is 100% accurate compared to an initial at least onedigital sequence. Further provided herein are methods, wherein the atleast one digital sequence comprises an amount of digital information ofat least 1 gigabyte. Further provided herein are methods, wherein the atleast one digital sequence comprises an amount of digital information ofat least t terabyte. Further provided herein are methods, wherein the atleast one digital sequence comprises an amount of digital information ofat least 1 petabyte.

Provided herein are methods for collection of information, the methodcomprising: (a) providing a structure comprising a surface, wherein thestructure comprises: a first plurality of polynucleotides havingpredetermined sequences collectively encoding for at least one nucleicacid sequence; and a second plurality of polynucleotides havingpredetermined sequences collectively encoding for the at least onenucleic acid sequence, wherein the first plurality of polynucleotidesand the second plurality of polynucleotides both extend from the surfaceand both encode for the same at least one nucleic acid sequence; (b)selectively separating a region of the structure comprising the firstplurality of polynucleotides and removing the first plurality ofpolynucleotides from the surface; and (c) sequencing and decrypting theat least one nucleic acid sequence to form at least one digital sequenceencoding for an item of information. Further provided herein aremethods, wherein a region of the structure comprising the firstplurality of polynucleotides comprises a cluster of channels or wells.Further provided herein are methods, wherein the structure is a rigidstructure. Further provided herein are methods, wherein the structure isa flexible structure. Further provided herein are methods, wherein aregion of the structure comprising only a remaining portion of thestructure lacking the first plurality of polynucleotides is spliced backtogether. Further provided herein are methods, wherein selectivelyremoving comprises application of force to a region of the structurecomprising the first plurality of polynucleotides. Further providedherein are methods, wherein the application of force is laminarpressure, capillary pressure, slip flow pressure, magnetic force,electrostatic force, peristaltic force, sound waves, vibrational force,centripetal force, centrifugal force, or any combination thereof.Further provided herein are methods, wherein the application of forcecomprises a conducting member, and an applied voltage potential betweenthe structure and the conducting member. Further provided herein aremethods, wherein the application of force comprises contacting thesurface of the structure with a rigid or flexible slip. Further providedherein are methods, wherein the application of force comprises apressure release or pressure nozzle. Further provided herein aremethods, wherein each polynucleotide of the first plurality ofnucleotides comprises at most 50) bases in length. Further providedherein are methods, wherein each polynucleotide of the first pluralityof nucleotides comprises at most 200 bases in length. Further providedherein are methods, wherein each polynucleotide of the second pluralityof nucleotides comprises at most 500 bases in length. Further providedherein are methods, wherein each polynucleotide of the second pluralityof nucleotides comprises at most 200 bases in length. Further providedherein are methods, wherein an amount of the item of information is atleast one gigabyte. Further provided herein are methods, wherein anamount of the item of information is at least one terabyte. Furtherprovided herein are methods, wherein an amount of the item ofinformation is at least one petabyte.

Provided herein are nucleic acid libraries, comprising a plurality ofpolynucleotides, wherein each of the polynucleotides comprises: (i) aplurality of coding regions, wherein each coding region is identical;and (ii) at least one non-coding region, wherein the at least onenon-coding region comprises a cleavage region; and wherein when theplurality of polynucleotides are sequenced, decrypted, and assembled toform a digital sequence, the digital sequence has greater than 90%accuracy compared to a preselected digital sequence. Further providedherein are nucleic acid libraries, wherein the cleavage region comprisesa restriction enzyme recognition site. Further provided herein arenucleic acid libraries, wherein the cleavage region comprises a lightsensitive nucleobase. Further provided herein are nucleic acidlibraries, further comprising application of a restriction enzyme,electromagnetic radiation, or a gaseous reagent to cleave at thecleavage region, thereby removing at least one of the plurality ofcoding regions. Further provided herein are nucleic acid libraries,wherein each coding region comprises 25 to 500 bases in length. Furtherprovided herein are nucleic acid libraries, wherein each coding regioncomprises 100 to 2000 bases in length. Further provided herein arenucleic acid libraries, wherein each non-coding region comprises 1 to 10bases in length. Further provided herein are nucleic acid libraries,wherein each non-coding region comprises at most 200 bases. Furtherprovided herein are nucleic acid libraries, wherein the plurality ofpolynucleotides comprises at least 100,000 polynucleotides. Furtherprovided herein are nucleic acid libraries, wherein the plurality ofpolynucleotides comprises at least 10 billion polynucleotides. Furtherprovided herein are nucleic acid libraries, wherein greater than 90% ofthe polynucleotides encode for a sequence that does not differ from apredetermined sequence. Further provided herein are nucleic acidlibraries, wherein a first non-coding region within each polynucleotidehas a different sequence than a second non-coding region within eachpolynucleotide. Further provided herein are nucleic acid libraries,wherein each non-coding region within each polynucleotide has adifferent sequence. Further provided herein are nucleic acid libraries,wherein a first cleavage region within each polynucleotide has adifferent sequence than a second cleavage region within eachpolynucleotide. Further provided herein are nucleic acid libraries,wherein each cleavage region within each polynucleotide has a differentsequence. Further provided herein are nucleic acid libraries, wherein anumber of cleavage regions within each polynucleotide is at least 1, 2,3, 4, or 5. Further provided herein are nucleic acid libraries, whereina sequence for the number of cleavage regions is different.

Provided herein are devices for storing information, the devicecomprising: (a) a structure having a surface; and (b) a plurality ofdiscrete regions on the surface for synthesizing a plurality ofpolynucleotides having predetermined sequences collectively encoding forat least one nucleic acid sequence, wherein each polynucleotidecomprises: (i) a plurality of coding regions, wherein each coding regionis identical; and (ii) at least one non-coding region, wherein the atleast one non-coding region comprises a cleavage region; and wherein theat least one nucleic acid sequence encodes for at least one item ofinformation.

Provided herein are devices for encrypting information, the devicecomprising: (a) a structure having a surface, wherein the surfacecomprises a plurality of non-identical markings; and (b) a plurality ofdiscrete regions on the surface for synthesizing a plurality ofpolynucleotides having predetermined sequences collectively encoding forat least one nucleic acid sequence, wherein the plurality ofpolynucleotides comprises at least 100,000 polynucleotides, and whereineach polynucleotide extends from the surface in a discrete regiondemarcated by one of the non-identical markings; and wherein the atleast one nucleic acid sequence encodes for at least one item ofinformation.

Provided herein are methods for storing information, the methodcomprising: (a) converting at least one item of information in the formof at least one digital sequence to at least one nucleic acid sequence;(b) synthesizing a plurality of polynucleotides having predeterminedsequences collectively encoding for the at least one nucleic acidsequence, wherein each polynucleotide comprises: (i) at least one codingsequence up to about 500 bases in length; and (ii) at least one bar codesequence, wherein the bar code sequence comprises sequence associatedwith the identity of the coding sequence; and (c) storing the pluralityof polynucleotides. Further provided herein are methods, wherein eachpolynucleotide comprises at least one coding sequence up to about 300bases in length. Further provided herein are methods, wherein theplurality of polynucleotides comprises at least about 100,000polynucleotides. Further provided herein are methods, wherein theplurality of polynucleotides comprises at least about 10 billionpolynucleotides. Further provided herein are methods, wherein greaterthan 90% of the polynucleotides encode for a sequence that does notdiffer from the predetermined sequence. Further provided herein aremethods, wherein the at least one item of information is textinformation, audio information or visual information.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates an exemplary workflow for nucleic acid-based datastorage.

FIGS. 2A-2C depict various polynucleotide sequence design schemes.

FIGS. 3A-3D depict various polynucleotide sequence design schemes.

FIGS. 4A-4B depict a barcode design scheme.

FIG. 5 illustrates a plate configured for polynucleotide synthesiscomprising 24 regions, or sub-fields, each having an array of 256clusters.

FIG. 6 illustrates a closer view of the sub-field in FIG. 5 having 16×16of clusters, each cluster having 121 individual loci.

FIG. 7 illustrates a detailed view of the cluster in FIG. 5, where thecluster has 121 loci.6

FIG. 8A illustrates a front view of a plate with a plurality ofchannels.

FIG. 8B illustrates a sectional view of plate with a plurality ofchannels.

FIGS. 9A-9B depict a continuous loop and reel-to-reel arrangements forflexible structures.

FIGS. 9C-9D depict schemas for release and extraction of synthesizedpolynucleotides.

FIGS. 10A-10C depict a zoom in of a flexible structure, having spots,channels, or wells, respectively.

FIG. 11A illustrates a zoom in of loci on a structure described herein.

FIGS. 11B-11C illustrate markings on structures described herein.

FIG. 12 illustrates a polynucleotide synthesis material depositiondevice.

FIG. 13 illustrates a polynucleotide synthesis workflow.

FIGS. 14A-14B illustrate a method for electrostatic deposition of apolynucleotide into a plurality of channels.

FIGS. 15A-15B illustrate an exemplary method for electrostatic transferof a polynucleotide from a plurality of channels.

FIGS. 16A-16B illustrate a method for transfer of a polynucleotide froma plurality of channels, through a slip mechanism.

FIGS. 17A-17B illustrate a method for transfer of a polynucleotide froma plurality of channels, through a pressure release mechanism.

FIG. 18 illustrates a method for transfer of a polynucleotide from aplurality of channels in a flexible structure, through a nozzlemechanism.

FIGS. 19A-19B illustrate a method for capture of a polynucleotide from aplurality of channels, through a pin.

FIGS. 20A-20B illustrate a method for electrostatic capture of apolynucleotide from a plurality of channels.

FIG. 21 illustrates a method for electrostatic containment of apolynucleotide from a plurality of channels into a receiving unit.

FIG. 22 illustrates a method for electrostatic containment of apolynucleotide from a plurality of channels into a receiving unit.

FIG. 23 illustrates an example of a computer system.

FIG. 24 is a block diagram illustrating architecture of a computersystem.

FIG. 25 is a diagram demonstrating a network configured to incorporate aplurality of computer systems, a plurality of cell phones and personaldata assistants, and Network Attached Storage (NAS).

FIG. 26 is a block diagram of a multiprocessor computer system using ashared virtual address memory space.

DETAILED DESCRIPTION OF THE INVENTION

There is a need for larger capacity storage systems as the amount ofinformation generated and stored is increasing exponentially.Traditional storage media have a limited capacity and requirespecialized technology that changes with time, requiring constanttransfer of data to new media, often at a great expense. A biomoleculesuch as a DNA molecule provides a suitable host for information storagein-part due to its stability over time and capacity for four bitinformation coding, as opposed to traditional binary information coding.Thus, large amounts of data are encoded in the DNA in a relativelysmaller amount of physical space than used by commercially availableinformation storage devices. Provided herein are methods to increase DNAsynthesis throughput through increased sequence density and decreasedturn-around time.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which these inventions belong.

Throughout this disclosure, numerical features are presented in a rangeformat. It should be understood that the description in range format ismerely for convenience and brevity and should not be construed as aninflexible limitation on the scope of any embodiments.

Accordingly, the description of a range should be considered to havespecifically disclosed all the possible subranges as well as individualnumerical values within that range to the tenth of the unit of the lowerlimit unless the context clearly dictates otherwise. For example,description of a range such as from 1 to 6 should be considered to havespecifically disclosed subranges such as from 1 to 3, from 1 to 4, from1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well asindividual values within that range, for example, 1.1, 2, 2.3, 5, and5.9. This applies regardless of the breadth of the range. The upper andlower limits of these intervening ranges may independently be includedin the smaller ranges, and are also encompassed within the invention,subject to any specifically excluded limit in the stated range. Wherethe stated range includes one or both of the limits, ranges excludingeither or both of those included limits are also included in theinvention, unless the context clearly dictates otherwise.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of any embodiment.As used herein, the singular forms “a,” “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising.” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof. As used herein, the term “and/or”includes any and all combinations of one or more of the associatedlisted items.

Unless specifically stated or obvious from context, as used herein, theterm “about” in reference to a number or range of numbers is understoodto mean the stated number and numbers+/−10% thereof, or 10% below thelower listed limit and 10% above the higher listed limit for the valueslisted for a range.

As used herein, the terms “preselected sequence”, “predefined sequence”or “predetermined sequence” are used interchangeably. The terms meanthat the sequence of the polymer is known and chosen before synthesis orassembly of the polymer. In particular, various aspects of the inventionare described herein primarily with regard to the preparation of nucleicacids molecules, the sequence of the oligonucleotide or polynucleotidebeing known and chosen before the synthesis or assembly of the nucleicacid molecules.

Provided herein are methods and compositions for production of synthetic(i.e. de novo synthesized or chemically synthesized) polynucleotides.Polynucleotides may also be referred to as oligonucleotides or oligos.Polynucleotide sequences described herein may be, unless statedotherwise, comprise DNA or RNA.

Nucleic Acid Based Information Storage

Provided herein are devices, compositions, systems and methods fornucleic acid-based information (data) storage. An exemplary workflow isprovided in FIG. 1. In a first step, a digital sequence encoding an itemof information (i.e., digital information in a binary code forprocessing by a computer) is received 101. An encryption 103 scheme isapplied to convert the digital sequence from a binary code to a nucleicacid sequence 105. A surface material for nucleic acid extension, adesign for loci for nucleic acid extension (aka, arrangement spots), andreagents for nucleic acid synthesis are selected 107. The surface of astructure is prepared for nucleic acid synthesis 108. De novopolynucleotide synthesis is performed 109. The synthesizedpolynucleotides are stored 111 and available for subsequent release 113,in whole or in part. Once released, the polynucleotides, in whole or inpart, are sequenced 115, subject to decryption 117 to convert nucleicsequence back to digital sequence. The digital sequence is thenassembled 119 to obtain an alignment encoding for the original item ofinformation.

Items of Information

Optionally, an early step of a DNA data storage process disclosed hereinincludes obtaining or receiving one or more items of information in theform of an initial code. Items of information include, withoutlimitation, text, audio and visual information. Exemplary sources foritems of information include, without limitation, books, periodicals,electronic databases, medical records, letters, forms, voice recordings,animal recordings, biological profiles, broadcasts, films, short videos,emails, bookkeeping phone logs, internet activity logs, drawings,paintings, prints, photographs, pixelated graphics, and software code.Exemplary biological profile sources for items of information include,without limitation, gene libraries, genomes, gene expression data, andprotein activity data. Exemplary formats for items of informationinclude, without limitation, .txt, .PDF, .doc, .docx, .ppt, .pptx, .xls,.xlsx, .rtf, .jpg, .gif, .psd, .bmp, .tiff, .png, and, mpeg. The amountof individual file sizes encoding for an item of information, or aplurality of files encoding for items of information, in digital formatinclude, without limitation, up to 1024 bytes (equal to 1 KB), 1024 KB(equal to 1 MB), 1024 MB (equal to 1 GB), 1024 GB (equal to 1 TB), 1024TB (equal to 1 PB), 1 exabyte, 1 zettabyte, 1 yottabyte, 1 xenottabyteor more. In some instances, an amount of digital information is at least1 gigabyte (GB). In some instances, the amount of digital information isat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000 or more than 1000 gigabytes. In some instances,the amount of digital information is at least 1 terabyte (TB). In someinstances, the amount of digital information is at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000 or more than 1000 terabytes. In some instances, the amount ofdigital information is at least 1 petabyte (PB). In some instances, theamount of digital information is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more than1000 petabytes.

Encryption

Binary Code Conversion

Generally, the initial code is digital information, typically in theform of binary code employed by a computer. General purpose computersare electronic devices reading “on” or “off” states, represented by thenumbers “0” and “1”. This binary code is application for computers toread multiple types of items of information. In binary arithmetic, thenumber two is written as the number 10. For example, “10” indicates “onetime the number, two and no more”. The number “3” is written as “11” tomean “one times two and one more.” The number “4” is written as “100,”the number “5” as “101” “six” as “110,” etc. An example of AmericanStandard Code II, (ASCII) for binary code is provided for the alphabetin lower and upper case in Table 1.

TABLE 1 ASCII Letter Code Binary a 97 1100001 b 98 1100010 c 99 1100011d 100 1100100 e 101 1100101 f 102 1100110 g 103 1100111 h 104 1101000 i105 1101001 j 106 1101010 k 107 1101011 l 108 1101100 m 109 1101101 n110 1101110 o 111 1101111 p 112 1110000 q 113 1110001 r 114 1110010 s115 1110011 t 116 1110100 u 117 1110101 v 118 1110110 w 119 1110111 x120 1111000 y 121 1111001 z 122 1111010 A 65 1000001 B 66 1000010 C 671000011 D 68 1000100 E 69 1000101 F 70 1000110 G 71 1000111 H 72 1001000I 73 1001001 J 74 1001010 K 75 1001011 L 76 1001100 M 77 1001101 N 781001110 O 79 1001111 P 80 1010000 Q 81 1010001 R 82 1010010 S 83 1010011T 84 1010100 U 85 1010101 V 86 1010110 W 87 1010111 X 88 1011000 Y 891011001 Z 90 1011010 0 chr(0) 00000000 1 chr(1) 00000001 2 chr(2)00000010 3 chr(3) 00000011 4 chr(4) 00000100 5 chr(5) 00000101 6 chr(6)00000110 7 chr(7) 00000111 8 chr(8) 00001000 9 chr(9) 00001001 10chr(10) 00001010 11 chr(11) 00001011 12 chr(12) 00001100 13 chr(13)00001101 14 chr(14) 00001110 15 chr(15) 00001111 16 chr(16) 00010000 17chr(17) 00010001 18 chr(18) 00010010 19 chr(19) 00010011 20 chr(20)00010100 21 chr(21) 00010101 22 chr(22) 00010110 23 chr(23) 00010111 24chr(24) 00011000 25 chr(25) 00011001 26 chr(26) 00011010 27 chr(27)00011011 28 chr(28) 00011100 29 chr(29) 00011101 30 chr(30) 00011110

Provided herein are methods for converting information in the form of afirst code, e.g., a binary sequence to a nucleic acid sequence. Theprocess may involve direct conversion from a base 2 code (i.e., binary)to a base code that is higher. Exemplary base codes include 2, 3, 4, 5,6, 7, 8, 9, 10 or more. Table 2 illustrates an exemplary alignmentbetween various base numbering schemes. A computer receiving machineinstructions for conversion, can automatically convert sequenceinformation from one code to another.

TABLE 2 Decimal 0 1 2 3 4 5 6 7 8 9 Quaternary 0 1 2 3 10 11 12 13 20 21Octal 0 1 2 3 4 5 6 7 10 11 Ternary 0 1 2 10 11 12 20 21 22 100 Binary 01 10 11 100 101 110 111 1000 1001

Canonical DNA is a base 4 coding system, having four differentnucleobases available: A, T, C or G (adenine, thymine, cytosine, andguanine). Thus, these 4 bases allow for a base 3 (using less than all),or a 4 base coding scheme. In addition, use of uracil (U), which isfound in RNA, provides a fifth base and allows for a base 5 codingscheme. In addition, modified nucleobase may be used for a nucleic acidbase coding greater than 4. Nucleobases that are not canonical DNAnucleobases or modified nucleobases include, without limitation, uracil,3-meA (3-methyladenine), hypoxanthine, 8-oxoG(7,8-dihydro-8-oxoguanine), FapyG, FapyA, Tg (thymine glycol), hoU(hydroxyuracil), hmU (hydroxymethyluracil), fU (formyluracil), hoC(hydroxycytosine), fC (formylcytosine), 5-meC (5-methylcytosine), 6-meG(O6-methylguanine), 7-meG (N7-methylguanine), sC (ethenocytosine), 5-caC(5-carboxylcytosine), 2-hA, cA (ethenoadenine), 5-fU (5-fluorouracil),3-meG (3-methylguanine), and isodialuric acid. Further provided hereinare coding schemes where machine instructions provide for conversion ofdigital information in the form of a binary sequence into anintermediate code prior to ultimately being converted to the finalnucleic acid sequence.

In some instances, to store data in a sequence of DNA, the informationis converted from the 1s and 0s of binary code into the code of A, T. G.and C bases of DNA. In some instances, items of information are firstencoded in a digital information form. In some cases, the binary code ofdigital information is converted into a biomolecule-based (e.g.,DNA-based) code while preserved the information that the coderepresents. This converted code (digital binary code to a biomoleculecode) is referred to herein as resulting in a “predetermined” sequencewith respect to the deposit of a biomolecule disclosed herein on asurface disclosed herein. The predetermined sequence may encode sequencefor a plurality of polynucleotides.

Nucleic Add Sequence

Provided herein are methods for designing a sequence for apolynucleotide described herein such that the nucleic acid sequenceencodes for at least part of an item of information. In some instances,each polynucleotide sequence has design features to facilitate withsequence alignment during subsequent assembly steps and also to providea means for error correction. In some arrangements, polynucleotidesequences are designed such that overlap exits between eachpolynucleotide sequence with another in the population. In someinstances, each polynucleotide sequence overlaps with a portion of justone other polynucleotide sequence. FIG. 2A. In an alternativearrangement, each polynucleotide sequence region overlaps with twosequences such that 2 copies are generated for each sequence within asingle polynucleotide, FIG. 2B. In yet another arrangement, eachpolynucleotide sequence region overlaps with more than two sequencessuch that 3 copies are generated for each sequence within a singlepolynucleotide, FIG. 2C. Sequences for polynucleotides described hereinmay encode for 10-2000, 10-500, 30-300, 50-250, or 75-200 bases inlength. In some instances, each of the polynucleotides sequence is atleast 10, 15, 20, 25, 30, 50, 100, 150, 200, 500 or more bases inlength.

In some arrangements, each polynucleotide sequence described herein isdesigned to comprise a plurality of coding regions and a plurality ofnon-coding regions, FIG. 3A. In such an arrangement, each coding region(e.g., 301, 303, 305) encodes for at least a portion of an item ofinformation. Optionally, each coding region in the same polynucleotideencodes for sequence from the same item of information, and anoverlapping scheme is optionally employed as described herein, FIG. 3B.In further instances, each coding region in the same polynucleotideencodes for the same sequence, FIGS. 3C-3D. Sequences forpolynucleotides described herein may encode for at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more codingregions. Sequences for polynucleotides described herein may encode forat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20 or more of the same coding region. In some instances, each of themultiple coding regions is 10-1000, 20-500, 30-300, 50-250, or 75-200bases in length. In some instances, each of the multiple coding regionsis 25-500, 25-200, 50-30, 50-200, 75-150, 10-2000, 20-1000 or 25-500bases in length. In some instances, each of the multiple coding regionsis at least 10, 15, 20, 25, 30, 50, 100, 150, 200 or more bases inlength. In some instances, each of the multiple coding regions is atleast 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 900, 1000, or more than 1000 bases. In someinstances, each of the multiple coding regions is at most 10, 50, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,900, 1000, or more than 1000 bases. In some instances, eachpolynucleotide comprises a tether region 311 linking the molecule to thesurface 302 of a structure.

In arrangements where multiple coding sequences are present in the samepolynucleotide, a cleavage region 307 is optionally present in betweeneach coding region. The cleavage region 307 may be present at thejunction between each coding region, or may be present within an adaptorregion having a string of sequence between each coding region. Acleavage region 307 may encode for a sequence feature, once synthesized,which will break from the strand subsequent to application of a cleavagesignal. The cleavage region 307 may encode for a restriction enzymerecognition site, a modified nucleic acid that is light sensitive andwill break under application of electromagnetic radiation (e.g.,oligodeoxynucleotide heteropolymers carrying base-sensitiveS-pivaloylthioethyl (t-Bu-SATE) phosphotriester linkages sensitive tolight wavelengths of >300 nm), or modified nucleic acid that issensitive to application of a certain chemical, e.g., Thymidine-succinylhexamide CED phosphoramidite (CLP-2244 from ChemGenes) which breakssubsequent to application of ammonia gas. Because the design of asequence to have a particular cleavage scheme may not be readilyapparent from sequencing synthesized polynucleotides, the cleavagescheme provides a means for adding a level of security to sequenceencoded by the synthesized nucleic acid library. Sequences forpolynucleotides described herein may encode for at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more cleavageregions. Sequences for polynucleotides described herein may encode forat least 1, 2, 3, 4, or 5 cleavage regions. In some instances, each ofthe cleavage region encodes for is 1-100, 1-50, 1-20, 1-10, 5-25, or5-30 bases in length. In some instances, each of the cleavage regionencodes for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 40, 50, 100 or more bases. In somearrangements, for each polynucleotide, each coding region is identicaland each cleavage region between each coding region is different. Forexample, a first cleavage region 307 is different from a second cleavageregion 309. In some arrangements, the cleavage region 307 closest to thesurface 302 is identical to the next distal cleavage region 307. In someinstances, each coding region is different from each of the other codingregion. For example, a first cleavage region 307 is different from asecond cleavage region 309 and from a third cleavage region 308.

Provided herein are polynucleotide sequences designed to comprise aplurality of coding regions and a plurality of non-coding regions,wherein the non-coding regions vary in length and number. For example,sequences for polynucleotides described herein may comprise at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 ormore non-coding regions. Sequences for polynucleotides described hereinmay comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20 or more of the same non-coding region. In someinstances, each of the multiple non-coding regions is 10-1000, 20-500,30-300, 50-250, or 75-200 bases in length. In some instances, each ofthe multiple non-coding regions is at least 1-100, 5-90, 10-80, 15-70,20-60, 25-50, or 30-40 bases in length. In some instances, each of themultiple non-coding regions is at least 10, 15, 20, 25, 30, 50, 100,150, 200 or more bases in length. In some instances, each of themultiple non-coding regions is at most 10, 15, 20, 25, 30, 50, 100, 150,200, or more bases in length. In some instances, the non-coding regionsare barcodes.

Barcodes are typically known nucleic acid sequences that allow somefeature of a polynucleotide with which the barcode is associated to beidentified. FIGS. 4A-4B provide an illustrative barcode arrangement. InFIG. 4A, each coding region for a first polynucleotide 301, a secondpolynucleotide 303, and a third polynucleotide 305, has the followingfeatures (from surface 302 outward); a tether region 302, a cleavageregion 307, an first primer binding region 401, a barcode region 403, acoding region 301, 303, 305, and a second primer binding region 404. Thepolynucleotides may be amplified with the use of primers that recognizethe first and/or second primer binding regions. Amplification may occurto polynucleotides attached to the surface or released from the surface(i.e., via cleavage at the cleavage region 307). After sequencing, thebarcode region 403, provides an indicator for identifying acharacteristic associated with the coding region. In some embodiments, abarcode comprises a nucleic acid sequence that when joined to a targetpolynucleotide serves as an identifier of the sample from which thetarget polynucleotide was derived. Barcodes can be designed at suitablelengths to allow sufficient degree of identification, e.g., at least 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, or morebases in length. Multiple barcodes, such as 2, 3, 4, 5, 6, 7, 8, 9, 10,or more barcodes, may be used on the same molecule, optionally separatedby non-barcode sequences. In some embodiments, barcodes are shorter than10.9, 8, 7, 6, 5, or 4 bases in length. In some embodiments, barcodesassociated with some polynucleotides are of different length thanbarcodes associated with other polynucleotides. In general, barcodes areof sufficient length and comprise sequences that are sufficientlydifferent to allow the identification of samples based on barcodes withwhich they are associated. In some arrangements, a barcode, and thesample source with which it is associated, can be identified accuratelyafter the mutation, insertion, or deletion of one or more bases in thebarcode sequence, such as the mutation, insertion, or deletion of 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more bases. In some embodiments, eachbarcode in a plurality of barcodes differ from every other barcode inthe plurality at least three base positions, such as at least 3, 4, 5,6, 7, 8, 9, 10, or more positions. Arrangements provided herein mayinclude bar codes sequence that correspond the nucleic acid sequence toencode sequence for a particular region of a digital sequence. Forexample, a barcode sequence may indicate where in a large file aparticular polynucleotide sequence encodes. In some instances, a barcodesequence may indicate which file a particular polynucleotide sequence isassociated with. In some instances, a barcode sequence includesinformation associated with the conversion scheme for a particularsequence, providing an added layer of security.

Provided herein are polynucleotide sequence design schemes where eachpolynucleotide sequence acid in a population is designed to have atleast one region in common amongst polynucleotide sequences in thatpopulation. For example, all polynucleotides in the same population maycomprise one or more primer regions. The design of sequence-specificprimer regions allows for the selection of polynucleotides to beamplified in selected batches from a large library of multiplepolynucleotides. Each polynucleotide sequence may comprise at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more primer binding sequences. Apopulation of polynucleotide sequence may comprise at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 25, 50, 100, 200, 500, 1000, 5000, 10000, 50000,100000 or more non-identical binding sequences. Primer binding sequencesmay comprise 5-100, 10-75, 7-60, 8-60, 10-50, or 10-40 bases in length.

Structures for Polynucleotide Synthesis

Provided herein are rigid or flexibles structures for polynucleotidesynthesis. In the case of rigid structures, provided herein are deviceshaving a structure (e.g., a plate) for the generation of a library ofpolynucleotides. An exemplary structure 500 is illustrated in FIG. 5,wherein the structure 500 has about the same size dimensions as astandard 96 well plate: 140 mm by 90 mm. The structure 500 comprisesclusters grouped in 24 regions or sub-fields 505, each sub-field 505comprising an array of 256 clusters 510. An expanded view of anexemplary sub-field 505 is shown in FIG. 6. In the expanded view of fourclusters (FIG. 6), a single cluster 510, has a Y axis cluster pitch(distance from center to center of adjacent clusters) of 1079.210 um or1142.694 um, and an X axis cluster pitch of 1125 um. An illustrativecluster 510 is depicted in FIG. 7, where the Y axis loci pitch (distancefrom center to center of adjacent loci) is 63.483 um, and an X axis locipitch is 75 um. The locus width at the longest part, e.g., diameter fora circular locus, is 50 um and the distance between loci is 24 um. Thenumber of loci 705 in the exemplary cluster in FIG. 7 is 121. The locimay be flat, wells, or channels. An exemplary channel arrangement isillustrated in FIGS. 8A-8B where a plate 805 is illustrated comprising amain channel 810 and a plurality of channels 815 connected to the mainchannel 810. The connection between the main channel 810 and theplurality of channels 815 provides for a fluid communication for flowpaths from the main channel 810 to the each of the plurality of channels815. A plate 805 described herein can comprise multiple main channels810. The plurality of channels 815 collectively forms a cluster withinthe main channel 810.

In the case of flexible structures, provided herein are devices whereinthe flexible structure comprises a continuous loop 901 wrapped aroundone or more fixed structures. e.g., a pair of rollers 903 or anon-continuous flexible structure 907 wrapped around separate fixedstructures, e.g., a pair reels 905. See FIGS. 9A-9B. In some instances,the structures comprise multiple regions for polynucleotide synthesis.An exemplary structure is illustrated in FIG. 9C where a plate comprisesdistinct regions 909 for polynucleotide synthesis. The distinct regions909 may be separated 911 by breaking or cutting. Each of the distinctregions may be further released, sequenced, decrypted, and read 913 orstored 915. An alternative structure is illustrated in FIG. 9D in whicha tape comprises distinct regions 917 for polynucleotide synthesis. Thedistinct regions 917 may be separated 919 by breaking or cutting. Eachof the distinct regions may be further released, sequenced, decrypted,and read 921 or stored 923. Provided herein are flexible structureshaving a surface with a plurality of loci for polynucleotide extension.FIGS. 10A-10C show a zoom in of the locus in the flexible structure.Each locus in a portion of the flexible structure 1001, may be asubstantially planar spot 1003 (e.g., flat), a channel 1005, or a well1007. In one exemplary arrangement, each locus of the structure has awidth of about 10 um and a distance between the center of each structureof about 21 um. See FIG. 11A. Loci may comprise, without limitation,circular, rectangular, tapered, or rounded shapes. Alternatively or incombination, the structures are rigid. In some instances, the rigidstructures comprise loci, channels, or wells for polynucleotidesynthesis.

In some instances, a channel described herein has a width to depth (orheight) ratio of 1 to 0.01, wherein the width is a measurement of thewidth at the narrowest segment of the microchannel. In some instances, achannel described herein has a width to depth (or height) ratio of 0.5to 0.01, wherein the width is a measurement of the width at thenarrowest segment of the microchannel. In some instances, a channeldescribed herein has a width to depth (or height) ratio of about 0.01,0.05, 0.1, 0.15, 0.16, 0.2, 0.5, or 1.

Described herein are structures comprising a plurality of discrete loci,channels, or wells for polynucleotide synthesis. In some instances,structures described herein are provided comprising a plurality ofchannels corresponding to a plurality of loci within a cluster, whereinthe height or depth of the channel is from about 5 um to about 500 um,from about 5 um to about 400 um, from about 5 um to about 300 um, fromabout 5 um to about 200 um, from about 5 um to about 100 um, from about5 um to about 50 um, or from about 10 um to about 50 um. In some cases,the height of a channel is less than 100 um, less than 80 um, less than60 urn, less than 40 um or less than 20 un. In some cases, channelheight is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400,500 um or more. In some instances, the height or depth of the channel isat least 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 7000, 800, 900,1000, or more than 1000 nm. In some instances, the height or depth ofthe channel is in a range of about 10 nm to about 1000 nm, about 25 nmto about 900 nm, about 50 nm to about 800 nm, about 75 nm to about 700nm, about 100 nm to about 600 nm, or about 200 nm to about 500.

In some instances, the width of a locus (e.g., substantially planarspot, well, or channel) is from about 0.1 um to about 50 um, from about0.5 um to about 500 um, from about 1 um to about 200 um, from about 1 umto about 100 um, from about 5 um to about 100 um, or from about 0.1 umto about 100 um, for example, about 90 um, 80 um, 70 um, 60 um, 50 um,40 um, 30 um, 20 um, 10 um, 5 um, 1 um or 0.5 um. In some instances, thewidth of a locus (e.g., microchannel) is less than about 100 um, 90 um,80 um, 70 um, 60 um, 50 um, 40 um, 30 um, 20 um or 10 um. In someinstances, the width of a locus is at least 10, 25, 50, 75, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, or more than 1000 nm. In someinstances, the width of a locus is in a range of about 10 nm to about1000 nm, about 25 nm to about 90) nm, about 50 nm to about 800 nm, about75 nm to about 700 nm, about 100 nm to about 600 nm, or about 200 nm toabout 500. In some instances, the distance between the center of twoadjacent loci is from about 0.1 um to about 500 um, 0.5 um to about 500um, from about 1 um to about 200 um, from about 1 um to about 100 um,from about 5 um to about 200 um, from about 5 um to about 100 um, fromabout 5 um to about 50 um, or from about 5 um to about 30 um, forexample, about 20 um. In some instances, the total width of a locus isabout 5 um, 10 um, 20 um, 30 um, 40 um, 50 um, 60 um, 70 um, 80 um, 90um, or 100 um. In some instances, the total width of a locus is about 1um to 100 um, 30 um to 100 um, or 50 um to 70 um.

In some instances, each locus supports the synthesis of a population ofpolynucleotides having a different sequence than a population ofpolynucleotides grown on another locus. Provided herein are surfaceswhich comprise at least 10, 100, 256, 500, 1000, 2000, 3000, 4000, 5000,6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 20000,30000, 40000, 5000 or more clusters. Provided herein are surfaces whichcomprise more than 2,000; 5,000; 10,000; 20,000; 30,000; 50,000;100,000; 200,000; 300,000; 40,000; 500,000; 600,000; 700,000; 800,000;900,000; 1,000,000; 5,000,000; or 10,000,000 or more distinct loci. Insome cases, each cluster includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 120, 130, 150, 200, 500 or more loci. Insome cases, each cluster includes 50 to 5 W, 50 to 200, 50 to 150, or100 to 150 loci. In some cases, each cluster includes 100 to 150 loci.In exemplary arrangements, each cluster includes 109, 121, 130 or 137loci.

Provided herein are loci having a width at the longest segment of 5 to100 um. In some cases, the loci have a width at the longest segment ofabout 30, 35, 40, 45, 50, 55 or 60 um. In some cases, the loci arechannels having multiple segments, wherein each segment has a center tocenter distance apart of 5 to 50 um. In some cases, the center to centerdistance apart for each segment is about 5, 10, 15, 20 or 25 um.

In some instances, the number of distinct polynucleotides synthesized onthe surface of a structure described herein is dependent on the numberof distinct loci available in the substrate. In some instances, thedensity of loci within a cluster of a substrate is at least or about 1locus per mm², 10 loci per mm², 25 loci per mm², 50 loci per mm², 65loci per mm², 75 loci per mm², 100 loci per mm², 130 loci per mm², 150loci per mm², 175 loci per mm², 200 loci per mm², 300 loci per mm², 400loci per mm², 500 loci per mm, 1,000 loci per mm² or more. In somecases, a substrate comprises from about 10 loci per mm² to about 500mm², from about 25 loci per mm² to about 400 mm², from about 50 loci permm² to about 500 mm², from about 100 loci per mm² to about 500 mm², fromabout 150 loci per mm² to about 500 mm², from about 10 loci per mm² toabout 250 mm², from about 50 loci per mm² to about 250 mm², from about10 loci per mm² to about 200 mm², or from about 50 loci per mm² to about200 mm². In some instances, the distance between the centers of twoadjacent loci within a cluster is from about 10 um to about 500 um, fromabout 10 um to about 200 um, or from about 10 urn to about 100 um. Insome cases, the distance between two centers of adjacent loci is greaterthan about 10 um, 20 um, 30 um, 40 um, 50 um, 60 um, 70 um, 80 um, 90 umor 100 um. In some cases, the distance between the centers of twoadjacent loci is less than about 200 um, 150 um, 100 um, 80 um, 70 um,60) um, 50 um, 40 um, 30 um, 20 um or 10 um. In some cases, the distancebetween the centers of two adjacent loci is less than about 10000 nm,8000 nm, 6000 nm, 4000 nm, 2000 nm 1000 nm, 800 nm, 600 nm, 400 nm, 200nm, ISO nm, 100 nm, 80 um, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm or10 nm. In some embodiments, each square meter of a structure describedherein allows for at least about 10⁷, 10⁸, 10¹⁰, 10¹¹ loci, where eachlocus supports one polynucleotide. In some embodiments, 10⁹polynucleotides are supported on less than about 6, 5, 4, 3, 2 or 1 m²of a structure described herein.

In some instances, a structure described herein provides support for thesynthesis of more than 2,000; 5,000; 10,000; 20,000, 30,000; 50,000,100,000; 200,000; 300,000; 400,000; 500,000; 60,000; 700,000; 800,000;900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000;2,000,000; 2,500,000; 3,000,000; 3,500,000, 4,000,000; 4,500,00,5,000,000; 10,000,000 or more non-identical polynucleotides. In somecases, the structure provides support for the synthesis of more than2,000, 5,000; 10,000; 20,000; 50,000; 100,000; 200,000; 300,000;400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000;1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000;3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; 10,000,000 ormore polynucleotides encoding for distinct sequences. In some instances,at least a portion of the polynucleotides have an identical sequence orare configured to be synthesized with an identical sequence. In someinstances, the structure provides a surface environment for the growthof polynucleotides having at least about 50, 60, 70, 75, 80, 85, 90, 95,100, 110, 120, 130, 140, 150, 160, 175, 200, 225, 250, 275, 300, 325,350, 375, 400, 425, 450, 475, 500 bases or more. In some arrangements,structures for polynucleotide synthesis described herein comprise sitesfor polynucleotide synthesis in a uniform arrangement.

In some instances, polynucleotides are synthesized on distinct loci of astructure, wherein each locus supports the synthesis of a population ofpolynucleotides. In some cases, each locus supports the synthesis of apopulation of polynucleotides having a different sequence than apopulation of polynucleotides grown on another locus. In some instances,the loci of a structure are located within a plurality of clusters. Insome instances, a structure comprises at least 10, 500, 1000, 2000,3000, 4000, 5000, 6000, 7000, 8000, 9000, 1000, 11000, 12000, 13000,14000, 15000, 20000, 30000, 40000, 50000 or more clusters. In someinstances, a structure comprises more than 2.000; 5.000; 10,000;100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000;900,000; 1,000,000; 1,100,000; 1,200,000; 1,300,000; 1,400,000;1,500,000; 1,600,000; 1,700,000; 1,800,000; 1,900,000; 2,000,000;300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000;1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000;2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; or10,000,000 or more distinct loci. In some cases, each cluster includes1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120,130, 150 or more loci. In some instances, each cluster includes 50 to500, 100 to 150, or 100 to 200 loci. In some instances, each clusterincludes 109, 121, 130 or 137 loci. In some instances, each clusterincludes 5, 6, 7, 8, 9, 10, 11 or 12 loci. In some instances,polynucleotides from distinct loci within one cluster have sequencesthat, when assembled, encode for a contiguous longer polynucleotide of apredetermined sequence.

Structure Size

In some instances, a structure described herein is about the size of astandard % well plate, for example between about 100 and 200 mm bybetween about 50 and 150 mm. In some instances, a structure describedherein has a diameter less than or equal to about 1000 mm, 500 mm, 450mm, 400 mm, 300 mm, 250 nm, 200 mm, 150 mm, 100 mm or 50 mm. In someinstances, the diameter of a substrate is between about 25 mm and 1000mm, between about 25 mm and about 800 mm, between about 25 mm and about600 mm, between about 25 mm and about 500 mm, between about 25 mm andabout 400 mm, between about 25 mm and about 300 mm, or between about 25mm and about 200. Non-limiting examples of substrate size include about300 mm, 200 mm, 150 mm, 130 mm, 100 mm, 76 mm, 51 mm and 25 mm. In someinstances, a substrate has a planar surface area of at least about 100mm²; 200 mm²; 500 mm²; 1,000 mm²; 2,000 mm²; 5,000 mm²; 10,000 mm²;12,000 mm²; 15,000 mm²; 20,000 mm² 30,000 mm², 40,000 mm 50,000 mm² ormore. In some instances, the thickness is between about 50 mm and about2000 mm, between about 50 mm and about 1000 mm, between about 100 mm andabout 1000 mm, between about 200 mm and about 1000 mm, or between about250 mm and about 1000 mm. Non-limiting examples thickness include 275mm, 375 mm, 525 mm, 625 mm, 675 mm, 725 mm, 775 mm and 925 mm. In somecases, the thickness of varies with diameter and depends on thecomposition of the substrate. For example, a structure comprisingmaterials other than silicon may have a different thickness than asilicon structure of the same diameter. Structure thickness may bedetermined by the mechanical strength of the material used and thestructure must be thick enough to support its own weight withoutcracking during handling. In some instances, a structure is more thanabout 1, 2, 3, 4, 5, 10, 15, 30, 40, 50 feet in any one dimension.

Materials

Provided herein are devices comprising a surface, wherein the surface ismodified to support polynucleotide synthesis at predetermined locationsand with a resulting low error rate, a low dropout rate, a high yield,and a high oligo representation. In some embodiments, surfaces ofdevices for polynucleotide synthesis provided herein are fabricated froma variety of materials capable of modification to support a de novopolynucleotide synthesis reaction. In some cases, the devices aresufficiently conducti, e, e.g., are able to form uniform electric fieldsacross all or a portion of the devices. Devices described herein maycomprise a flexible material. Exemplary flexible materials include,without limitation, modified nylon, unmodified nylon, nitrocellulose,and polypropylene. Devices described herein may comprise a rigidmaterial. Exemplary rigid materials include, without limitation, glass,fuse silica, silicon, silicon dioxide, silicon nitride, plastics (forexample, polytetrafluoroethylene, polypropylene, polystyrene,polycarbonate, and blends thereof, and metals (for example, gold,platinum). Devices disclosed herein may be fabricated from a materialcomprising silicon, polystyrene, agarose, dextran, cellulosic polymers,polyacrylamides, polydimethylsiloxane (PDMS), glass, or any combinationthereof. In some cases, devices disclosed herein is manufactured with acombination of materials listed herein or any other suitable materialknown in the art.

Devices described herein may comprise material having a range of tensilestrength. Exemplary materials having a range of tensile strengthsinclude, but are not limited to, nylon (70 MPa), nitrocellulose (1.5MPa), polypropylene (40 MPa), silicon (268 MPa), polystyrene (40 MPa),agarose (1-10 MPa), polyacrylamide (1-10 MPa), polydimethylsiloxane(PDMS) (3.9-10.8 MPa). Solid supports described herein can have atensile strength from 1 to 300, 1 to 40, 1 to 10, 1 to 5, or 3 to 11MPa. Solid supports described herein can have a tensile strength ofabout 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 20, 25, 40, 50, 60, 70,80, 90, 100, 150, 200, 250, 270, or more MPa. In some instances, adevice described herein comprises a solid support for polynucleotidesynthesis that is in the form of a flexible material capable of beingstored in a continuous loop or reel, such as a tape or flexible sheet.

Young's modulus measures the resistance of a material to elastic(recoverable) deformation under load. Exemplary materials having a rangeof Young's modulus stiffness include, but are not limited to, nylon (3GPa), nitrocellulose (1.5 GPa), polypropylene (2 GPa), silicon (050GPa), polystyrene (3 GPa), agarose (0-10 GPa), polyacrylamide (1-10GPa), polydimethylsiloxane (PDMS) (1-10 GPa). Solid supports describedherein can have a Young's moduli from 1 to 500, 1 to 40, 1 to 10, 1 to5, or 3 to 11 GPa. Solid supports described herein can have a Young'smoduli of about 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 20, 25, 40, 50,60, 70, 80, 90, 100, 150, 200, 250, 400, 500 GPa or more. As therelationship between flexibility and stiffness are inverse to eachother, a flexible material has a low Young's modulus and changes itsshape considerably under load. In some instances, a solid supportdescribed herein has a surface with a flexibility of at least nylon.

In some cases, devices disclosed herein comprises a silicon dioxide baseand a surface layer of silicon oxide. Alternatively, the devices mayhave a base of silicon oxide. Surface of the devices provided here maybe textured, resulting in an increase overall surface area forpolynucleotide synthesis. Devices disclosed herein may comprise at least5%, 10% , 25%, 50%, 80%, 90%, 95%, or 99% silicon. Devices disclosedherein may be fabricated from a silicon on insulator (SOI) wafer.

The structure may be fabricated from a variety of materials, suitablefor the methods and compositions of the invention described herein. Incertain embodiments, the materials from which the substrates/solidsupports of the comprising the invention are fabricated exhibit a lowlevel of oligonucleotide binding. In some situations, material that aretransparent to visible and/or UV light can be employed. Materials thatare sufficiently conductive, e.g. those that can form uniform electricfields across all or a portion of the substrates/solids supportdescribed herein, can be utilized. In some embodiments, such materialsmay be connected to an electric ground. In some cases, the substrate orsolid support can be heat conductive or insulated. The materials can bechemical resistant and heat resistant to support chemical or biochemicalreactions such as a series of oligonucleotide synthesis reaction. Forflexible materials, materials of interest can include: nylon, bothmodified and unmodified, nitrocellulose, polypropylene, and the like.

For rigid materials, specific materials of interest include: glass; fusesilica; silicon, plastics (for example polytetraflouroethylene,polypropylene, polystyrene, polycarbonate, and blends thereof, and thelike); metals (for example, gold, platinum, and the like). The structurecan be fabricated from a material selected from the group consisting ofsilicon, polystyrene, agarose, dextran, cellulosic polymers,polyacrylamides, polydimethylsiloxane (PDMS), and glass. Thesubstrates/solid supports or the microstructures, reactors therein maybe manufactured with a combination of materials listed herein or anyother suitable material known in the art.

The term “flexible” is used herein to refer to a structure that iscapable of being bent, folded or similarly manipulated without breakage.In some cases, a flexible structure is bent at least 30 degrees around aroller. In some cases, a flexible structure is bent at least 180 degreesaround a roller. In some cases, a flexible structure is bent at least270 degrees around a roller. In some instances, a flexible structure isbent about 360 degrees around a roller. In some cases, the roller isless than about 10 cm, 5 cm, 3 cm, 2 cm or 1 cm in radius. In someinstances, the flexible structure is bent and straightened repeatedly ineither direction at least 100 times without failure (for example,cracking) or deformation at 20° C. In some instances, a flexiblestructure described herein has a thickness that is amenable to rolling.In some cases, the thickness of the flexible structure described hereinis less than about 50 mm, 10 mm, 1 mm, or 0.5 mm.

Exemplary flexible materials for structure described herein include,without limitation, nylon (unmodified nylon, modified nylon, clearnylon), nitrocellulose, polypropylene, polycarbonate, polyethylene,polyurethane, polystyrene, acetal, acrylic, acrylonitrile, butadienestyrene (ABS), polyester films such as polyethylene terephthalate,polymethyl methacrylate or other acrylics, polyvinyl chloride or othervinyl resin, transparent PVC foil, transparent foil for printers,Poly(methyl methacrylate) (PMMA), methacrylate copolymers, styrenicpolymers, high refractive index polymers, fluorine-containing polymers,polyethersulfone, polyimides containing an alicyclic structure, rubber,fabric, metal foils, and any combination thereof. Various plasticizersand modifiers may be used with polymeric substrate materials to achieveselected flexibility characteristics.

Flexible structures described herein may comprise a plastic material. Insome instances, the flexible structure comprises a thermoplasticmaterial. Non-limiting examples of thermoplastic materials includeacrylic, acrylonitrile butadiene styrene, nylon, polylactic acid,polybenzimidazole, polycarbonate, polyether sulfone, polyetheretherketone, polyetherimide, polyethylene, polyphenylene oxide, polyphenylenesulfide, polypropylene, polystyrene, polyvinyl chloride, andpolytetrafluoroethylene. In some embodiments, the substrate comprises athermoplastic material in the polyaryletherketone (PEAK) family.Non-limiting examples of PEAK thermoplastics include polyetherketone(PEK), polyetherketoneketone (PEKK), poly(ether ether ketone ketone)(PEEKK), polyether ether ketone (PEEK), andpolyetherketoneetherketoneketone (PEKEKK). In some instances, theflexible structure comprises a thermoplastic material compatible withtoluene. In some instances, the flexibility of the plastic material isincreased by the addition of a plasticizer. An example of a plasticizeris an ester-based plasticizer, such as phthalate. Phthalate plasticizersinclude bis(2-ethylhexyl) phthalate (DEHP), diisononly phthalate (DINP),di-n-butyl phthalate (DnBP, DBP), butyl benzyl phthalate (BBzP),diisodecyl phthalate (DIDP), dioctyl phthalate (DOP, DnOP), diisooctylphthalate (DIOP), diethyl phthalate (DEP), diisobutyl phthalate (DIBP),and di-n-hexyl phthalate. In some instances, modification of thethermoplastic polymer through copolymerization or through the additionof non-reactive side chains to monomers before polymerization alsoincreases flexibility.

Provided herein are flexible structures which may further comprise afluoroelastomer. Materials having about 80% fluoroelastomers aredesignated as FKMs. Fluoroelastomers include perfluoro-elastomers(FFKMs) and tetrafluoroethylene/propylene rubbers (FEPM).Fluoroelastomers have five known types. Type 1 FKMs are composed ofvinylidene fluoride (VDF) and hexafluoropropylene (HFP) and theirfluorine content typically is around 66% by weight. Type 2 FKMs arecomposed of VDF, HFP, and tetrafluoroethylene (TFE) and typically havebetween about 68% and 69% fluorine. Type 3 FKMs are composed of VDF,TFE, and perfluoromethylvinylether (PMVE) and typically have betweenabout 62% and 68% fluorine. Type 4 FKMs are composed of propylene, TFE,and VDF and typically have about 67% fluorine. Type 5 FKMs are composedof VDF, HFP, TFE. PMVE, and ethylene.

In some instances, a substrate disclosed herein comprises a computerreadable material. Computer readable materials include, withoutlimitation, magnetic media, reel-to-reel tape, cartridge tape, cassettetape, flexible disk, paper media, film, microfiche, continuous tape(e.g., a belt) and any media suitable for storing electronicinstructions. In some cases, the substrate comprises magneticreel-to-reel tape or a magnetic belt. In some instances, the substratecomprises a flexible printed circuit board.

Structures described herein may be transparent to visible and/or UVlight. In some instances, structures described herein are sufficientlyconductive to form uniform electric fields across all or a portion of astructure. In some instances, structures described herein are heatconductive or insulated. In some instances, the structures are chemicalresistant and heat resistant to support a chemical reaction such as apolynucleotide synthesis reaction. In some embodiments, the substrate ismagnetic. In some instances, the structures comprise a metal or a metalalloy.

Structures for polynucleotide synthesis may be over 1, 2, 5, 10, 30, 50or more feet long in any dimension. In the case of a flexible structure,the flexible structure is optionally stored in a wound state, e.g., in areel. In the case of a large rigid structure. e.g., greater than 1 footin length, the rigid structure can be stored vertically or horizontally.

Encryption Key Markings on the Structure's Surface

Provided herein are structures having markings 1101 wherein the markingsprovide information relating to the source item of informationassociated with a nearby population of polynucleotides, an encryptionscheme for decrypting the sequence of the nearby population ofpolynucleotides, the copy number for the nearby population ofpolynucleotides, or any combination thereof. See, e.g., FIGS. 11B-11C.The markings may be visible to the naked eye, or visible under amagnified view using a microscope. In some instances, the markings onthe surface are only visible after a treatment condition to expose themarking, such as a heat, chemical or light treatment (e.g., UV or IRlight to illuminate the marking). An example ink developed by heatincludes, without limitation, cobalt chloride, (which turns blue whenheated). Example inks developed by chemical reaction include, withoutlimitation, phenolphthalein, copper sulfate, lead(II) nitrate,cobalt(II) chloride, and cerium oxalate developed by manganese sulfateand hydrogen peroxide.

Surface Preparation

Provided herein are methods to support the immobilization of abiomolecule on a substrate, where a surface of a structure describedherein comprises a material and/or is coated with a material thatfacilitates a coupling reaction with the biomolecule for attachment. Toprepare a structure for biomolecule immobilization, surfacemodifications may be employed that chemically and/or physically alterthe substrate surface by an additive or subtractive process to changeone or more chemical and/or physical properties of a substrate surfaceor a selected site or region of the surface. For example, surfacemodification involves (1) changing the wetting properties of a surface.(2) functionalizing a surface, i.e. providing, modifying or substitutingsurface functional groups, (3) defunctionalizing a surface, i.e.removing surface functional groups, (4) otherwise altering the chemicalcomposition of a surface. e.g., through etching, (5) increasing ordecreasing surface roughness, (6) providing a coating on a surface,e.g., a coating that exhibits wetting properties that are different fromthe wetting properties of the surface, and/or (7) depositingparticulates on a surface. In some instances, the surface of a structureis selectively functionalized to produce two or more distinct areas on astructure, wherein at least one area has a different surface or chemicalproperty that another area of the same structure. Such propertiesinclude, without limitation, surface energy, chemical termination,surface concentration of a chemical moiety, and the like.

In some instances, a surface of a structure disclosed herein is modifiedto comprise one or more actively functionalized surfaces configured tobind to both the surface of the substrate and a biomolecule, therebysupporting a coupling reaction to the surface. In some instances, thesurface is also functionalized with a passive material that does notefficiently bind the biomolecule, thereby preventing biomoleculeattachment at sites where the passive functionalization agent is bound.In some cases, the surface comprises an active layer only definingdistinct loci for biomolecule support.

In some embodiments, the surface is contacted with a mixture offunctionalization groups which are in any different ratio. In someembodiments, a mixture comprises at least 2, 3, 4, 5 or more differenttypes of functionalization agents. In some cases, the ratio of the atleast two types of surface functionalization agents in a mixture isabout 1:1, 1:2, 1:5, 1:10, 2:10, 3:10, 4:10, 5:10, 6:10, 7:10, 8:10,9:10, or any other ratio to achieve a desired surface representation oftwo groups. In some embodiments, desired surface tensions,wettabilities, water contact angles, and/or contact angles for othersuitable solvents are achieved by providing a substrate surface with asuitable ratio of functionalization agents. In some cases, the agents ina mixture are chosen from suitable reactive and inert moieties, thusdiluting the surface density of reactive groups to a desired level fordownstream reactions. In some embodiments, the mixture offunctionalization reagents comprises one or more reagents that bind to abiomolecule and one or more reagents that do not bind to a biomolecule.Therefore, modulation of the reagents allows for the control of theamount of biomolecule binding that occurs at a distinct area offunctionalization.

In some instances, a method for substrate functionalization comprisesdeposition of a silane molecule onto a surface of a substrate. Thesilane molecule may be deposited on a high energy surface of thesubstrate. In some instances the high surface energy region includes apassive functionalization reagent. Methods described herein provide fora silane group to bind the surface, while the rest of the moleculeprovides a distance from the surface and a free hydroxyl group at theend to which a biomolecule attaches. In some instances, the silane is anorganofunctional alkoxysilane molecule. Non-limiting examples oforganofunctional alkoxysilane molecules includedimethylchloro-octodecyl-silane, methyldichloro-octodecyl-silane,trichloro-octodecyl-silane, and trimethyl-oxodecyl-silane,triethyl-octodecyl-silane. In some instances, the silane is an aminosilane. Examples of amino silanes include, without limitation,11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane.(3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane,glycidyloxypropyl/trimethoxysilane andN-(3-triethoxysilylpropyl)-4-hydroxybutyramide. In some instances, thesilane comprises 11-acetoxyundecyltriethoxysilane,n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane.(3-aminopropyl)triethoxysilane, glycidyloxypropyl/trimethoxysilane,N-(3-triethoxysilylpropyl)-4-hydroxy butyramide, or any combinationthereof. In some instances, an active functionalization agent comprises11-acetoxyundecyltriethoxysilane. In some instances, an activefunctionalization agent comprises n-decyltriethoxysilane. In some cases,an active functionalization agent comprisesglycidyloxypropyltriethoxysilane (GOPS). In some embodiments, the silaneis a fluorosilane. In some embodiments, the silane is a hydrocarbonsilane. In some cases, the silane is 3-iodo-propyltrimethoxysilane. Insome cases, the silane is octylchlorosilane.

In some embodiments, silanization is performed on a surface throughself-assembly with organofunctional alkoxysilane molecules. Theorganofunctional alkoxysilanes are classified according to their organicfunctions. Non-limiting examples of siloxane functionalizing reagentsinclude hydroxyalkyl siloxanes (silylate surface, functionalizing withdiborane and oxidizing the alcohol by hydrogen peroxide), diol(dihydroxyalkyl) siloxanes (silylate surface, and hydrolyzing to diol),aminoalkyl siloxanes (amines require no intermediate functionalizingstep), glycidoxysilanes (3-glycidoxypropyl-dimethyl-ethoxysilane,glycidoxy-trimethoxysilane), mercaptosilanes(3-mercaptopropyl-trimethoxysilane, 3-4epoxycyclohexyl-ethyltrimethoxysilane or3-mercaptopropyl-methyl-dimethoxysilane),bicyclohepthenyl-trichlorosilane, butyl-aldehydr-trimethoxysilane, ordimeric secondary aminoalkyl siloxanes. Exemplary hydroxyalkyl siloxanesinclude allyl trichlorochlorosilane turning into 3-hydroxypropyl, or7-oct-1-enyl trichlorochlorosilane turning into 8-hydroxyoctyl. The diol(dihydroxyalkyl) siloxanes include glycidyl trimethoxysilane-derived(2,3-dihydroxypropyloxy)propyl (GOPS). The aminoalkyl siloxanes include3-aminopropyl trimethoxysilane turning into 3-aminopropyl(3-aminopropyl-triethoxysilane, 3-aminopropyl-diethoxy-methylsilane,3-aminopropyl-dimethyl-ethoxysilane, or 3-aminopropyl-trimethoxysilane).In some cases, the dimeric secondary aminoalkyl siloxanes is bis(3-trimethoxysilylpropyl) amine turning into bis(silyloxylpropyl)amine.

Active functionalization areas may comprise one or more differentspecies of silanes, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moresilanes. In some cases, one of the one or more silanes is present in thefunctionalization composition in an amount greater than another silane.For example, a mixed silane solution having two silanes comprises a99:1, 98:2, 97:3, 96:4, 95:5, 94:6, 93:7, 92:8, 91:9, 90:10, 89:11,88:12, 87:13, 86:14, 85:15, 84:16, 83:17, 82:18, 81:19, 80:20, 75:25,70:30, 65:35, 60:40, 55:45 ratio of one silane to another silane. Insome instances, an active functionalization agent comprises11-acetoxyundecyltriethoxysilane and n-decyltriethoxysilane. In someinstances, an active functionalization agent comprises11-acetoxyundecyltriethoxysilane and n-decyltriethoxysilane in a ratiofrom about 20:80 to about 1:99, or about 10:90 to about 2:98, or about5:95.

In some instances, functionalization comprises deposition of afunctionalization agent to a structure by any deposition technique,including, but not limiting to, chemical vapor deposition (CVD), atomiclayer deposition (ALD plasma enhanced CVD (PECVD), plasma enhanced ALD(PEALD), metal organic CVD (MOCVD), hot wire CVD (HWCVD), initiated CVD(iCVD), modified CVD (MCVD), vapor axial deposition (VAD), outside vapordeposition (OVD), physical vapor deposition (e.g., sputter deposition,evaporative deposition), and molecular layer deposition (MLD).

Any step or component in the following functionalization process beomitted or changed in accordance with properties desired of the finalfunctionalized substrate. In some cases, additional components and/orprocess steps are added to the process workflows embodied herein. Insome instances, a substrate is first cleaned, for example, using apiranha solution. An example of a cleaning process includes soaking asubstrate in a piranha solution (e.g., 90% H₂SO₄, 10% H₂O₂) at anelevated temperature (e.g., 120° C.) and washing (e.g., water) anddrying the substrate (e.g., nitrogen gas). The process optionallyincludes a post piranha treatment comprising soaking the piranha treatedsubstrate in a basic solution (e.g., NH₄OH) followed by an aqueous wash(e.g., water). In some instances, a surface of a structure is plasmacleaned, optionally following the piranha soak and optional post piranhatreatment. An example of a plasma cleaning process comprises an oxygenplasma etch. In some instances, the surface is deposited with an activefunctionalization agent following by vaporization. In some instances,the substrate is actively functionalized prior to cleaning, for example,by piranha treatment and/or plasma cleaning.

The process for surface functionalization optionally comprises a resistcoat and a resist strip. In some instances, following active surfacefunctionalization, the substrate is spin coated with a resist, forexample, SPR™ 3612 positive photoresist. The process for surfacefunctionalization, in various instances, comprises lithography withpatterned functionalization. In some instances, photolithography isperformed following resist coating. In some instances, afterlithography, the surface is visually inspected for lithography defects.The process for surface functionalization, in some instances, comprisesa cleaning step, whereby residues of the substrate are removed, forexample, by plasma cleaning or etching. In some instances, the plasmacleaning step is performed at some step after the lithography step.

In some instances, a surface coated with a resist is treated to removethe resist, for example, after functionalization and/or afterlithography. In some cases, the resist is removed with a solvent, forexample, with a stripping solution comprising N-methyl-2-pyrrolidone. Insome cases, resist stripping comprises sonication or ultrasonication. Insome instances, a resist is coated and stripped, followed by activefunctionalization of the exposed areas to create a desired differentialfunctionalization pattern.

In some instances, the methods and compositions described herein relateto the application of photoresist for the generation of modified surfaceproperties in selective areas, wherein the application of thephotoresist relies on the fluidic properties of the surface defining thespatial distribution of the photoresist. Without being bound by theory,surface tension effects related to the applied fluid may define the flowof the photoresist. For example, surface tension and/or capillary actioneffects may facilitate drawing of the photoresist into small structuresin a controlled fashion before the resist solvents evaporate. In someinstances, resist contact points are pinned by sharp edges, therebycontrolling the advance of the fluid. The underlying structures may bedesigned based on the desired flow patterns that are used to applyphotoresist during the manufacturing and functionalization processes. Asolid organic layer left behind after solvents evaporate may be used topursue the subsequent steps of the manufacturing process. Structures maybe designed to control the flow of fluids by facilitating or inhibitingwicking effects into neighboring fluidic paths. For example, a structureis designed to avoid overlap between top and bottom edges, whichfacilitates the keeping of the fluid in top structures allowing for aparticular disposition of the resist. In an alternative example, the topand bottom edges overlap, leading to the wicking of the applied fluidinto bottom structures. Appropriate designs may be selected accordingly,depending on the desired application of the resist.

In some instances, a structure described herein has a surface thatcomprises a material having thickness of at least or at least about 0.1nm, 0.5 nm, 1 nm, 2 nm, 5 nm, 10 nm or 25 nm that comprises a reactivegroup capable of binding nucleosides. Exemplary include, withoutlimitation, glass and silicon, such as silicon dioxide and siliconnitride. In some cases, exemplary surfaces include nylon and PMMA.

In some instances, electromagnetic radiation in the form of UV light isused for surface patterning. In some instances, a lamp is used forsurface patterning, and a mask mediates exposure locations of the UVlight to the surface. In some instances, a laser is used for surfacepatterning, and a shutter opened/closed state controls exposure of theUV light to the surface. The laser arrangement may be used incombination with a flexible structure that is capable of moving. In suchan arrangement, the coordination of laser exposure and flexiblestructure movement is used to create patterns of one or more agentshaving differing nucleoside coupling capabilities.

Material Deposition Systems

Provided herein are systems and devices for the deposition and storageof biomolecules on a structure described herein. In some embodiments,the biomolecules are polynucleotides that store encoded information intheir sequences. In some embodiments, the system comprises a surface ofa structure to support biomolecule attachment and/or a device forapplication of a biomolecule to the surface of the substrate. In anexample, the device for biomolecule application is a polynucleotidesynthesizer. In some embodiments, the system comprises a device fortreating the substrate with a fluid, for example, a flow cell. In someembodiments, the system comprises a device for moving the substratebetween the application device and the treatment device. For instanceswhere the substrate is a reel-to-reel tape, the system may comprise twoor more reels that allow for access of different portions of thesubstrate to the application and optional treatment device at differenttimes.

A first example of a polynucleotide material deposition system forpolynucleotide synthesis is shown in FIG. 12. The system includes amaterial deposition device that moves in the X-Y direction to align withthe location of the substrate. The material deposition device can alsomove in the Z direction to seal with the substrate, forming a resolvedreactor. A resolved reactor is configured to allow for the transfer offluid, including polynucleotides and/or reagents, from the substrate toa capping element and/or vice versa. As shown in FIG. 12, fluid may passthrough either or both the substrate and the capping element andincludes, without limitation, coupling reagents, capping reagents,oxidizers, de-blocking agents, acetonitrile and nitrogen gas. Examplesof devices that are capable of high resolution droplet depositioninclude the printhead of inkjet printers and laser printers. The devicesuseful in the systems and methods described herein achieve a resolutionfrom about 100 dots per inch (DPI) to about 50,000 DPI; from about 100DPI to about 20,000 DPI; from about 100 DPI to about 10,000 DPI; fromabout 100 DPI to about 5,000 DPI; from about 1,000 DPI to about 20,000DPI; or from about 1,000 DPI to about 10,000 DPI. In some instances, thedevices have a resolution at least about 1,000; 2,000; 3,000; 4,000;5,000; 10,000; 12,000 DPI, or 20,000 DPI. The high resolution depositionperformed by the device is related to the number and density of eachnozzle that corresponds to a feature of the substrate.

An exemplary process workflow for de novo synthesis of a polynucleotideon a substrate using a polynucleotide synthesizer is shown in FIG. 13.Droplets comprising polynucleotide synthesis reagents are released fromthe material deposition device to the substrate in a stepwise manner,wherein the material deposition device has a piezo ceramic material andelectrodes to convert electrical signals into a mechanical signal forreleasing the droplets. The droplets are released to specific locationson the surface of the substrate one nucleobase at a time to generate aplurality of synthesized polynucleotides having predetermined sequencesthat encode data. In some cases, the synthesized polynucleotides arestored on the substrate. Nucleic acid reagents may be deposited on thesubstrate surface in a non-continuous, or drop-on-demand method.Examples of such methods include the electromechanical transfer method,electric thermal transfer method, and electrostatic attraction method.In the electromechanical transfer method, piezoelectric elementsdeformed by electrical pulses cause the droplets to be ejected. In theelectric thermal transfer method, bubbles are generated in a chamber ofthe device, and the expansive force of the bubbles causes the dropletsto be ejected. In the electrostatic attraction method, electrostaticforce of attraction is used to eject the droplets onto the substrate. Insome cases, the drop frequency is from about 5 KHz to about 500 KHz;from about 5 KHz to about 1(0) KHz; from about 10 KHz to about 500 KHz;from about 10 KHz to about 100 KHz; or from about 50 KHz to about 500KHz. In some cases, the frequency is less than about 500 KHz, 200 KHz,100 KHz, or 50 KHz.

The size of the droplets dispensed correlates to the resolution of thedevice. In some instances, the devices deposit droplets of reagents atsizes from about 0.01 pl to about 20 pl, from about 0.01 pl to about 10pl, from about 0.01 pl to about 1 pl, from about 0.01 pl to about 0.5pl, from about 0.01 pl to about 0.01 pl, or from about 0.05 pl to about1 pl. In some instances, the droplet size is less than about 1 pl, 0.5pl, 0.2 pl, 0.1 pl, or 0.05 pl. The size of droplets dispensed by thedevice is correlated to the diameters of deposition nozzles, whereineach nozzle is capable of depositing a reagent onto a feature of thesubstrate. In some instances, a deposition device of a polynucleotidesynthesizer comprises from about 100 to about 10,000 nozzles; from about100 to about 5,000 nozzles; from about 100 to about 3,000 nozzles; fromabout 500 to about 10,000 nozzles; or from about 100 to about 5,000nozzles. In some cases, the deposition device comprises greater than1,000; 2,000; 3,000; 4,000; 5,000; or 10,000 nozzles. In some instances,each material deposition device comprises a plurality of nozzles, whereeach nozzle is optionally configured to correspond to a feature on asubstrate. Each nozzle may deposit a reagent component that is differentfrom another nozzle. In some instances, each nozzle deposits a dropletthat covers one or more features of the substrate. In some embodiments,one or more nozzles are angled. In some embodiments, multiple depositiondevices are stacked side by side to achieve a fold increase inthroughput. In some cases, the gain is 2×, 4×, 8× or more. An example ofa deposition device is Samba Printhead (Fujifilm). A Samba Printhead maybe used with the Samba Web Administration Tool (SWAT).

The number of deposition sites may be increased by using and rotatingthe same deposition device by a certain degree or saber angle. Byrotating the deposition device, each nozzle is jetted with a certainamount of delay time corresponding to the saber angle. Thisunsynchronized jetting creates a cross talk among the nozzles.Therefore, when the droplets are jetting at a certain saber angledifferent from 0 degrees, the droplet volume from the nozzle could bedifferent.

In some arrangements, the configuration of a polynucleotide synthesissystem allows for a continuous polynucleotide synthesis process thatexploits the flexibility of a substrate for traveling in a reel-to-reeltype process. This synthesis process operates in a continuous productionline manner with the substrate travelling through various stages ofpolynucleotide synthesis using one or more reels to rotate the positionof the substrate. In an exemplary embodiment, a polynucleotide synthesisreaction comprises rolling a substrate: through a solvent bath, beneatha deposition device for phosphoramidite deposition, through a bath ofoxidizing agent, through an acetonitrile wash bath, and through adeblock bath. Optionally, the tape is also traversed through a cappingbath. A reel-to-reel type process allows for the finished product of asubstrate comprising synthesized polynucleotides to be easily gatheredon a take-up reel, where it can be transported for further processing orstorage.

In some arrangements, polynucleotide synthesis proceeds in a continuousprocess as a continuous flexible tape is conveyed along a conveyor beltsystem. Similar to the reel-to-reel type process, polynucleotidesynthesis on a continuous tape operates in a production line manner,with the substrate travelling through various stages of polynucleotidesynthesis during conveyance. However, in a conveyor belt process, thecontinuous tape revisits a polynucleotide synthesis step without rollingand unrolling of the tape, as in a reel-to-reel process, in somearrangements, polynucleotide synthesis steps are partitioned into zonesand a continuous tape is conveyed through each zone one or more times ina cycle. For example, a polynucleotide synthesis reaction may comprise(1) conveying a substrate through a solvent bath, beneath a depositiondevice for phosphoramidite deposition, through a bath of oxidizingagent, through an acetonitrile wash bath, and through a block bath in acycle; and then (2) repeating the cycles to achieve synthesizedpolynucleotides of a predetermined length. After polynucleotidesynthesis, the flexible substrate is removed from the conveyor beltsystem and, optionally, rolled for storage. Rolling may be around areel, for storage.

In an exemplary arrangement, a flexible substrate comprisingthermoplastic material is coated with nucleoside coupling reagent. Thecoating is patterned into loci such that each locus has diameter ofabout 10 um, with a center-to-center distance between two adjacent lociof about 21 um. In this instance, the locus size is sufficient toaccommodate a sessile drop volume of 0.2 pl during a polynucleotidesynthesis deposition step. In some cases, the locus density is about 2.2billion loci per m² (1 locus/441×10⁻¹² m²). In some cases, a 4.5 m²substrate comprise about 10 billion loci, each with a 10 um diameter.

A material deposition device described herein may comprises about 2,048nozzles that each deposit about 100,000 droplets per second at 1nucleobase per droplet. For each deposition device, at least about1.75×10¹³ nucleobases are deposited on the substrate per day. In someinstances, 100 to 500 nucleobase polynucleotides are synthesized. Insome cases, 200 nucleobase polynucleotides are synthesized. Optionally,over 3 days, at a rate of about 1.75×10¹³ bases per day, at least about262.5×10⁹ polynucleotides are synthesized.

In some arrangements, a device for application of one or more reagentsto a substrate during a synthesis reaction is configured to depositreagents and/or nucleotide monomers for nucleoside phosphoramidite basedsynthesis. Reagents for polynucleotide synthesis include reagents forpolynucleotide extension and wash buffers. As non-limiting examples, thedevice deposits cleaning reagents, coupling reagents, capping reagents,oxidizers, de-blocking agents, acetonitrile, gases such as nitrogen gas,and any combination thereof. In addition, the device optionally depositsreagents for the preparation and/or maintenance of substrate integrity.In some embodiments, the polynucleotide synthesizer deposits a drophaving a diameter less than about 200 um, 100 um, or 50 um in a volumeless than about 1000, 500, 100, 50, or 20 pl. In some cases, thepolynucleotide synthesizer deposits between about 1 and 10000, 1 and5000, 100 and 5000, or 1000 and 5000 droplets per second.

In some arrangement, during polynucleotide synthesis, the substrate ispositioned within and/or sealed within a flow cell. The flow cell mayprovide continuous or discontinuous flow of liquids such as thosecomprising reagents necessary for reactions within the substrate, forexample, oxidizers and/or solvents. The flow cell may provide continuousor discontinuous flow of a gas, such as nitrogen, for drying thesubstrate typically through enhanced evaporation of a volatilesubstrate. A variety of auxiliary devices are useful to improve dryingand reduce residual moisture on the surface of the substrate. Examplesof such auxiliary drying devices include, without limitation, a vacuumsource, depressurizing pump and a vacuum tank. In some cases, apolynucleotide synthesis system comprises one or more flow cells, suchas 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 and one or more substrates, such as2, 3, 4, 5, 6, 7, 8, 9, 10 or 20. In some cases, a flow cell isconfigured to hold and provide reagents to the substrate during one ormore steps in a synthesis reaction. In some embodiments, a flowcellcomprises a lid that slides over the top of a substrate and can beclamped into place to form a pressure tight seal around the edge of thesubstrate. An adequate seal includes, without limitation, a seal thatallows for about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 atmospheres ofpressure. In some cases, the lid of the flow cell is opened to allow foraccess to an application device such as a polynucleotide synthesizer. Insome cases, one or more steps of a polynucleotide synthesis method areperformed on a substrate within a flow cell, without the transport ofthe substrate.

In some arrangements, a device for treating a substrate with a fluidcomprises a spray bar. Nucleotide monomers may be applied onto asubstrate surface then a spray bar sprays the substrate surface with oneor more treatment reagents using spray nozzles of the spray bar. In somearrangements, the spray nozzles are sequentially ordered to correlatewith different treatment steps during polynucleotide synthesis. Thechemicals used in different process steps may be changed in the spraybar to readily accommodate changes in a synthesis method or betweensteps of a synthesis method. In some embodiments, the spray barcontinuously sprays a given chemistry on a surface of a substrate as thesubstrate moves past the spray bar. In some cases, the spray bardeposits over a wide area of a substrate, much like the spray bars usedin lawn sprinklers. In some embodiments, the spray bar nozzles arepositioned to provide a uniform coat of treatment material to a givenarea of a substrate.

In some embodiments, a polynucleotide synthesis system comprises one ormore elements useful for downstream processing of synthesizedpolynucleotides. As an example, the system comprises a temperaturecontrol element such as a thermal cycling device. In some embodiments,the temperature control element is used with a plurality of resolvedreactors to perform nucleic acid assembly such as PCA and/or nucleicacid amplification such as PCR.

De Novo Polynucleotide Synthesis

Provided herein are systems and methods for synthesis of a high densityof polynucleotides on a substrate in a short amount of time. In someinstances, the substrate is a flexible substrate. In some instances, atleast about 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, or 10¹⁵ bases are synthesizedin one day. In some instances, at least about 10×10⁸, 10×10⁹, 10×10¹⁰,10×10¹¹, or 10×10¹² polynucleotides are synthesized in one day. In somecases, each polynucleotide synthesized comprises at least about 20, 50,100, 200, 300, 400 or 500 nucleobases. In some cases, these bases aresynthesized with a total average error rate of less than about 1 in 100;200; 300; 400; 500; 1000; 2000; 5000; 10000; 15000; 20000 bases. In someinstances, these error rates are for at least 50%, 60%, 70%, 80%, 90%,95%, 98%, 99%, 99.5%, or more of the polynucleotides synthesized. Insome instances, these at least 90%, 95%, 98%, 99%, 99.5%, or more of thepolynucleotides synthesized do not differ from a predetermined sequencefor which they encode. In some instances, the error rate for synthesizedpolynucleotides on a substrate using the methods and systems describedherein is less than about 1 in 200. In some instances, the error ratefor synthesized polynucleotides on a substrate using the methods andsystems described herein is less than about 1 in 1,000. In someinstances, the error rate for synthesized polynucleotides on a substrateusing the methods and systems described herein is less than about 1 in2,000. In some instances, the error rate for synthesized polynucleotideson a substrate using the methods and systems described herein is lessthan about 1 in 3,000. In some instances, the error rate for synthesizedpolynucleotides on a substrate using the methods and systems describedherein is less than about 1 in 5,000. Individual types of error ratesinclude mismatches, deletions, insertions, and/or substitutions for thepolynucleotides synthesized on the substrate. The term “error rate”refers to a comparison of the collective amount of synthesizedpolynucleotide to an aggregate of predetermined polynucleotidesequences. In some instances, synthesized polynucleotides disclosedherein comprise a tether of 12 to 25 bases. In some embodiments, thetether comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50 or more bases.

A suitable method for polynucleotide synthesis on a substrate of thisdisclosure is a phosphoramidite method comprising the controlledaddition of a phosphoramidite building block, i.e. nucleosidephosphoramidite, to a growing polynucleotide chain in a coupling stepthat forms a phosphite triester linkage between the phosphoramiditebuilding block and a nucleoside bound to the substrate. In someinstances, the nucleoside phosphoramidite is provided to the substrateactivated. In some instances, the nucleoside phosphoramidite is providedto the substrate with an activator. In some instances, nucleosidephosphoramidites are provided to the substrate in a 1.5, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50,60, 70, 80, 90, 100-fold excess or more over the substrate-boundnucleosides. In some instances, the addition of nucleosidephosphoramidite is performed in an anhydrous environment, for example,in anhydrous acetonitrile. Following addition and linkage of anucleoside phosphoramidite in the coupling step, the substrate isoptionally washed. In some embodiments, the coupling step is repeatedone or more additional times, optionally with a wash step betweennucleoside phosphoramidite additions to the substrate. In someinstances, a polynucleotide synthesis method used herein comprises 1, 2,3 or more sequential coupling steps. Prior to coupling, in many cases,the nucleoside bound to the substrate is de-protected by removal of aprotecting group, where the protecting group functions to preventpolymerization. A common protecting group is 4,4′-dimethoxytrityl (DMT).

Following coupling, phosphoramidite polynucleotide synthesis methodsoptionally comprise a capping step. In a capping step, the growingpolynucleotide is treated with a capping agent. A capping step generallyserves to block unreacted substrate-bound 5′-OH groups after couplingfrom further chain elongation, preventing the formation ofpolynucleotides with internal base deletions. Further, phosphoramiditesactivated with 1H-tetrazole often react, to a small extent, with the O6position of guanosine. Without being bound by theory, upon oxidationwith I₂/water, this side product, possibly via O6-N7 migration,undergoes depurination. The apurinic sites can end up being cleaved inthe course of the final deprotection of the oligonucleotide thusreducing the yield of the full-length product. The O6 modifications maybe removed by treatment with the capping reagent prior to oxidation withI₂/water. In some embodiments, inclusion of a capping step duringpolynucleotide synthesis decreases the error rate as compared tosynthesis without capping. As an example, the capping step comprisestreating the substrate-bound polynucleotide with a mixture of aceticanhydride and 1-methylimidazole. Following a capping step, the substrateis optionally washed.

Following addition of a nucleoside phosphoramidite, and optionally aftercapping and one or more wash steps, the substrate bound growing nucleicacid may be oxidized. The oxidation step comprises oxidizing thephosphite triester into a tetracoordinated phosphate triester, aprotected precursor of the naturally occurring phosphate diesterinternucleoside linkage. In some instances, oxidation of the growingpolynucleotide is achieved by treatment with iodine and water,optionally in the presence of a weak base such as a pyridine, lutidine,or collidine. Oxidation is sometimes carried out under anhydrousconditions using tert-Butyl hydroperoxide or(1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO). In some methods, acapping step is performed following oxidation. A second capping stepallows for substrate drying, as residual water from oxidation that maypersist can inhibit subsequent coupling. Following oxidation, thesubstrate and growing polynucleotide is optionally washed. In someembodiments, the step of oxidation is substituted with a sulfurizationstep to obtain oligonucleotide phosphorothioates, wherein any cappingsteps can be performed after the sulfurization. Many reagents arecapable of the efficient sulfur transfer, including, but not limited to,3-(Dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-3-thione. DDTT,3H-1,2-benzodithiol-3-one 1,1-dioxide, also known as Beaucage reagent,and N,N,N′N′-Tetraethylthiuram disulfide (TETD).

In order for a subsequent cycle of nucleoside incorporation to occurthrough coupling, a protected 5′ end of the substrate bound growingpolynucleotide must be removed so that the primary hydroxyl group canreact with a next nucleoside phosphoramidite. In some instances, theprotecting group is DMT and deblocking occurs with trichloroacetic acidin dichloromethane. Conducting detritylation for an extended time orwith stronger than recommended solutions of acids may lead to increaseddepurination of solid support-bound oligonucleotide and thus reduces theyield of the desired full-length product. Methods and compositionsdescribed herein provide for controlled deblocking conditions limitingundesired depurination reactions. In some instances, the substrate boundpolynucleotide is washed after deblocking. In some cases, efficientwashing after deblocking contributes to synthesized polynucleotideshaving a low error rate.

Methods for the synthesis of polynucleotides on the substrates describedherein typically involve an iterating sequence of the following steps:application of a protected monomer to a surface of a substrate featureto link with either the surface, a linker or with a previouslydeprotected monomer; deprotection of the applied monomer so that it canreact with a subsequently applied protected monomer; and application ofanother protected monomer for linking. One or more intermediate stepsinclude oxidation and/or sulfurization. In some instances, one or morewash steps precede or follow one or all of the steps.

In some embodiments, polynucleotides are synthesized with photolabileprotecting groups, where the hydroxyl groups generated on the surfaceare blocked by photolabile-protecting groups. When the surface isexposed to UV light, such as through a photolithographic mask, a patternof free hydroxyl groups on the surface may be generated. These hydroxylgroups can react with photoprotected nucleoside phosphoramidites,according to phosphoramidite chemistry. A second photolithographic maskcan be applied and the surface can be exposed to LV light to generatesecond pattern of hydroxyl groups, followed by coupling with5′-photoprotected nucleoside phosphoramidite. Likewise, patterns can begenerated and oligomer chains can be extended. Without being bound bytheory, the lability of a photocleavable group depends on the wavelengthand polarity of a solvent employed and the rate of photocleavage may beaffected by the duration of exposure and the intensity of light. Thismethod can leverage a number of factors such as accuracy in alignment ofthe masks, efficiency of removal of photo-protecting groups, and theyields of the phosphoramidite coupling step. Further, unintended leakageof light into neighboring sites can be minimized. The density ofsynthesized oligomer per spot can be monitored by adjusting loading ofthe leader nucleoside on the surface of synthesis.

The surface of the substrate that provides support for polynucleotidesynthesis may be chemically modified to allow for the synthesizedpolynucleotide chain to be cleaved from the surface. In some instances,the polynucleotide chain is cleaved at the same time as thepolynucleotide is deprotected. In some cases, the polynucleotide chainis cleaved after the polynucleotide is deprotected. In an exemplaryscheme, a trialkoxysilyl amine such as (CH3CH2O)3Si—(CH2)2-NH2 isreacted with surface SiOH groups of a substrate, followed by reactionwith succinic anhydride with the amine to create an amide linkage and afree OH on which the nucleic acid chain growth is supported. Cleavageincludes gas cleavage with ammonia or methylamine. In some instances,once released from the surface, polynucleotides are assembled intolarger nucleic acids that are sequenced and decoded to extract storedinformation.

Assembly

Polynucleotides may be designed to collectively span a large region of apredetermined sequence that encodes for information. In some instances,larger polynucleotides are generated through ligation reactions to jointhe synthesized polynucleotides. One example of a ligation reaction ispolymerase chain assembly (PCA). In some instances, at least of aportion of the polynucleotides are designed to include an appendedregion that is a substrate for universal primer binding. For PCAreactions, the presynthesized polynucleotides include overlaps with eachother (e.g., 4, 20, 40 or more bases with overlapping sequence). Duringthe polymerase cycles, the polynucleotides anneal to complementaryfragments and then are filled in by polymerase. Each cycle thusincreases the length of various fragments randomly depending on whichpolynucleotides find each other. Complementarity amongst the fragmentsallows for forming a complete large span of double-stranded DNA. In somecases, after the PCA reaction is complete, an error correction step isconducted using mismatch repair detecting enzymes to remove mismatchesin the sequence. Once larger fragments of a target sequence aregenerated, they can be amplified. For example, in some cases, a targetsequence comprising 5′ and 3′ terminal adapter sequences is amplified ina polymerase chain reaction (PCR) which includes modified primers thathybridize to the adapter sequences. In some cases, the modified primerscomprise one or more uracil bases. The use of modified primers allowsfor removal of the primers through enzymatic reactions centered ontargeting the modified base and/or gaps left by enzymes which cleave themodified base pair from the fragment. What remains is a double-strandedamplification product that lacks remnants of adapter sequence. In thisway, multiple amplification products can be generated in parallel withthe same set of primers to generate different fragments ofdouble-stranded DNA.

Error correction may be performed on synthesized polynucleotides and/orassembled products. An example strategy for error correction involvessite-directed mutagenesis by overlap extension PCR to correct errors,which is optionally coupled with two or more rounds of cloning andsequencing. In certain embodiments, double-stranded nucleic acids withmismatches, bulges and small loops, chemically altered bases and/orother heteroduplexes are selectively removed from populations ofcorrectly synthesized nucleic acids. In some embodiments, errorcorrection is performed using proteins/enzymes that recognize and bindto or next to mismatched or unpaired bases within double-strandednucleic acids to create a single or double-strand break or to initiate astrand transfer transposition e, ent. Non-limiting examples ofproteins/enzymes for error correction include endonucleases (T7Endonuclease I, E. coli Endonuclease V, T4 Endonuclease VII, mung beannuclease, Cell. E. coli Endonuclease IV, UVDE), restriction enzymes,glycosylases, ribonucleases, mismatch repair enzymes, resolvases,helicases, ligases, antibodies specific for mismatches, and theirvariants. Examples of specific error correction enzymes include T4endonuclease 7, T7 endonuclease 1, S1, mung bean endonuclease, MutY,MutS, MutH, Mu(L, cleavase, CELI, and HINF1. In some cases, DNAmismatch-binding protein MutS (Therma aquaticus) is used to removefailure products from a population of synthesized products. In someembodiments, error correction is performed using the enzyme Correctase.In some cases, error correction is performed using SURVEYOR endonuclease(Transgenomic), a mismatch-specific DNA endonuclease that scans forknown and unknown mutations and polymorphisms for heteroduplex DNA.

Release, Extraction and Assembly

Provided herein are method and devices for replicable informationstorage. In some instances, multiple copies of the same coding region,the polynucleotide, the same cluster, the same portion of a structurecomprising polynucleotides, or the entire structure comprisingpolynucleotides are synthesized. Where multiple copies of the samepolynucleotide are synthesized, each of the polynucleotides may areattached to distinct regions of the surface. The distinct regions may beseparated by breaking or cutting. Alternatively, each of thepolynucleotides may be present at a locus in the form of a spot, well orchannel and individually accessible. For example, contacting the locuswith a cleavage reagent and then water would free one copy of thepolynucleotide while leaving the other copies intact. Similarly,cleavage of polynucleotides in an entire region or over an entire plateallows for accessing a fraction of a replicate population. Replicatepopulations may exist in separated reels, plates, belts, and the like.In the case of a flexible material, such as a tape, a replicate regionmay be cut and the remaining regions of the tape may be spliced backtogether. Alternatively, nucleic acid information of the synthesized andstored polynucleotides may be obtained by performing amplification ofpolynucleotides attached to the surface of the structure using primersand a DNA polymerase.

In some instances, an aqueous or gaseous transfer media is depositedonto one or a plurality of channels in a structure to transfer thepolynucleotides from the structure to a receiving unit. For example, atransfer media may pass through a channel in the structure to adhere to,collect and transfer a polynucleotide from a channel in the structure toa receiving unit. In some instance, a charge conducting feature and anapplied voltage are employed to attract or repel a transfer media to orthrough a channel in the structure. In some instances, a slip isemployed to direct a transfer media into a channel in the structure. Insome cases a pressure release is employed to direct a transfer mediainto or through a channel in the structure. In some cases a nozzle isemployed to form a localized area of high pressure which forces atransfer media into or through a channel in the structure. In someinstances, a pin is employed to transfer a polynucleotide from a channelin the structure to a container to a receiving unit. In such instances,the pin may comprise agents to facilitate transfer media adhesion. Insome cases a charge conducting feature is employed to attract or repel atransfer media to or through a channel in a structure, by forming avoltage potential between the conducting feature and the structure. Insome cases, a pipette tip, or other capillary flow inducing structure,is used to transfer the fluid and polynucleotides via capillary flow. Insome instances, a container comprises one or more compartments that eachreceives a portion of the transfer media, and the one or morepolynucleotides therein, emitted from a single respective channel. Insome instances, the container comprises a single compartment thatreceives one or more portions of the transfer media, each containing oneor more polynucleotides therein, emitted from a one or more structurechannels.

Referring to FIGS. 14A and 14B, a polynucleotide 1417 is transferredfrom a channel 1415 in a structure 1405 through the deposition of anaqueous or gaseous transfer media 1419, which adheres to apolynucleotide 1417, and wherein one or more interconnected conductorplates 1420 and a power unit 1422 direct the transfer media 1419 to theone or more channels respectively. In this arrangement, a series of oneor more interconnected conductor plates 1420 are each located above, andsurround, the proximal edge a respective channel 1415, and wherein avoltage potential imparted by a power unit 1422 between theinterconnected conductor plates 1420 and the structure 1405, attractsthe transfer media 1419 to the proximal opening of one or more channels1415. As such, an exemplary method of attracting the transfer media 1419to the proximal opening of the one or more channels 1415 in thisinstance comprises: depositing a transfer media 1419 into a main channel1410 of a structure 1405, and applying a voltage potential between theinterconnected conductor plates 1420 and the structure 1405, via a powerunit 1422. Further, in this case, the transfer media 1419 may contain apositive or negative charge which reacts to an electrostatic or magneticfield or a potential difference created by the power unit 1422, as itpasses through the structure 1405 and the channels 1415. Additionally,the electrostatic properties of the one or more conductor plates 1420and the structure 1405 can be tuned to optimize the transfer of thepolynucleotide 1417 within the transfer media 1419 through a channel1415 in the structure. Finally, a nonconductive separator may bepositioned between the structure 1405 and the one or more conductorplates 1420, to tune or optimize the electrostatic or magnetic field orthe potential difference formed therein. Further, this case mayadditionally employ hydrophilic or hydrophobic structures on one or morefaces of the main channel 1410 or on the interconnected conductor plates1420 to more efficiently direct the transfer media 1419 into thechannels 1415.

Referring to FIGS. 15A and 15B, a polynucleotide 1517 is transferredfrom a channel 1515 in a structure 1505, through the deposition of anaqueous or gaseous transfer media 1519 which adheres to one or morepolynucleotides 1517, and wherein the transfer media 1519 is attractedthrough the one or more channels 1515 by one or more conducting sheets1524 and a power unit 1522. In this arrangement, a conducting sheet 1524below and surrounding the distal edge of a channel 1515, and a powerunit 1522, are employed to attract the transfer media 1519 from theproximal opening of a channel 1515, see FIG. 15A, to the distal openingof that channel 1515, see FIG. 15B. As such, an exemplary method ofattracting the transfer media 1519 to the distal opening of a channel1515 in this instance comprises: applying a voltage potential between aconducting sheet 1524 and the structure 1505, via a power unit 1522.Further, in this case, the transfer media 1519 may contain a positive ornegative charge which reacts to an electrostatic or magnetic field or apotential difference created by the power unit 1522, as it passesthrough the structure 1505, and the one or more conducting sheets 1524.Additionally, the electrostatic properties of the one or more conductingsheets 1524 and the structure 1505 can be tuned to optimize the transferof the polynucleotides 1517 in the transfer media 1519 through a channel1515 in the structure 1505. A nonconductive separator may be positionedbetween the structure 1505 and the one or more conducting sheets 1524,to tune or optimize the electrostatic or magnetic field or the potentialdifference formed therein.

Referring to FIGS. 16A and 16B, a polynucleotide 1617 is transferredfrom a channel 1615 in a structure 1605, through the deposition of anaqueous or gaseous transfer media 1619 which adheres to a polynucleotide1617, and wherein a slip 1630, in flush contact with the surface of, andpositioned at an acute angle of attack 1632 relative to, a stationaryplate structure 1605 or a moving non-continuous flexible structure, isemployed to direct a transfer media into to the channels of thestructure. In this arrangement, a slip 1630 is employed to direct thetransfer media 1619 from the proximal opening of the one or morechannels 1615, see FIG. 16A, to the distal opening of the respectivechannel 1615, see FIG. 16B. As such, an exemplary method of directingthe transfer media 1619 through one or more channels 1615 in thisinstance comprises: translating or rotating the one or more slips 1630relative to the structure 1605. In these instances the acute angle ofattack 1632 may be equal to about 10°, 20°, 30°, 40°, 50°, 60°, 70° orabout 80°. In some cases, a single slip 1630 or a rigid assembly of oneor more slips 1630 is employed to direct the transfer media 1619 throughthe one or more channels 1615. In some cases, the relative velocitybetween the slip 1630 and the structure 1605 is up to about 1centimeter/second. In some cases, the relative velocity between the slip1630 and the structure 1605 is more than 1 centimeter/second. In somecases, the relative angular velocity between the slip 1630 relative tothe structure 1605 is up to about 1 rotation/second. In some cases, therelative angular velocity between the slip 1630 and the structure 1605is more than 1 rotation/second. In some cases, the slip 1630 can contortto partially enter the channel 1615. Finally, in this instance, the slip1630 may be composed of any waterproof material comprising plastic,rubber, wood, metal, glass, fiberglass, carbon fiber or any combinationthereof.

The case wherein a polynucleotide 1717 is transferred from a channel1715 in a structure 1705, through the deposition of an aqueous orgaseous transfer media 1719 which adheres to a polynucleotide 1717, andwherein an applied pressure 1740 within a gas or fluid, and a pressurerelease 1742 are employed to force the transfer media 1719 through achannel 1715 in the structure 1705, is displayed in FIGS. 17A and 17B.In this instance, a pressure release 1742 block the applied pressure1740, thus forming a pressure differential between distal edge a channel1715, and the distal face a pressure release 1742, see FIG. 17A, which,when released by the opening of a pressure release 1742, forces thetransfer media 1719 through a channel, see FIG. 17B. In some cases, asingle pressure release 1742 is employed to direct the transfer media1719 through one or more channels 1715 at once. As such, an exemplarymethod of directing the transfer media 1719 through a channel 1715 inthis instance comprises: forming an applied pressure 1740 within the gasor fluid, and translating or rotating a pressure release 1742 relativeto the structure 1705. In some cases, the relative velocity between theone or more pressure releases 1742 and the structure 705 is up to about1 centimeter/second. In some cases, the relative velocity between theone or more pressure releases 1742 and the structure 1705 is more than 1centimeter/second. In some arrangements, the relative rotationalvelocity between the one or more pressure releases 1742 and thestructure 1705 is up to about 1 rotation/second. In some cases, therelative rotational velocity between the one or more pressure releases1742 and the structure 1705 is more than 1 rotation/second. In someinstances the pressure differential within the gas or fluid surroundingthe structure 1705, created by the applied pressure 1740 is less than 1atm. In some instances the pressure differential within the gas or fluidsurrounding the structure 1705, created by the applied pressure 1740 ismore than 1 atm.

Referring to FIG. 18, a polynucleotide 1817 is transferred from achannel 1815 in a moving non-continuous flexible structure 1807, throughthe deposition of an aqueous or gaseous transfer media 1819 whichadheres a polynucleotide 1817, and wherein a nozzle 1844 and an appliedpressure 1840, are employed to force a transfer media 1819 through achannel 1815 in the structure 1807. As such, an exemplary method ofdirecting the transfer media 1819 through a channel 1815 in thisinstance comprises: translating the continuous flexible structure 1807,about a roller 1803 such that a channel 1815 is aligned below a nozzle1844, and triggering a nozzle 1844 to direct an applied pressure 1840towards a channel 1815. In some instances, the pressure differentialwithin the gas or fluid surrounding the structure 1807 imparted by anozzle 1844 is less than 1 atm. In some instances the pressuredifferential within the gas or fluid surrounding the structure 1807imparted by a nozzle 1844 is more than 1 atm.

Referring to FIGS. 19A and 19B, a polynucleotide 1917 is transferredfrom a channel 1915 in a structure 1905, through the deposition of anaqueous or gaseous transfer media 1919, and wherein a pin 1950 adheresto the transfer media 1919, and a polynucleotide 1917 within, to removethe transfer media 1919 from a channel 1915 in the structure 1905 isshown in FIGS. 19A and 19B. In this instance, the pin 1950 contacts andattracts the transfer media 1919, see FIG. 19A, wherein the attractionof the transfer media 1919 to the pin 1950 is greater than the transfermedia's 1919 attraction to the distal edge of a channel 1915, andwherein a relative vertical motion between the pin 1950 and thestructure 1905 dislocates the transfer media 1919 from the structure1905, see FIG. 19B. In such instances, the pin 1950 may comprisefeatures to facilitate transfer media adhesion comprising hydrophilic orgas-philic structures or coatings, or a binding chemical coating. Insome instances, the pin 1950 is comprised of any hard materialcomprising metal, plastic, rubber, carbon fiber, wood, fiberglass or anycombination thereof. In other instances, the pin 1950 is comprised of aconductive material capable of conducting an electric, electrostatic ormagnetic charge or field to attract the transfer media 1919. In somecases, the relative velocity between the pin 1950 and the structure 1905is up to about 1 centimeter/second. In some cases, the relative velocitybetween the pin 1950 and the structure 1905 is more than 1centimeter/second.

Referring to FIGS. 20A and 20B, a polynucleotide 2017 is transferredfrom a channel 2015 in a structure 2005 through the deposition of anaqueous or gaseous transfer media 2019, and wherein the transfer media2019 is repelled from a channel in the structure 2005 to a receivingunit 2060, by a voltage applied from a power unit 2022 to a conductingsheet 2024. In this instance, a conducting sheet 2024 below andsurrounding the distal edge of a channel 2015, and a power unit 2022 areemployed to repel the transfer media 2019 from the distal opening of achannel 2015, see FIG. 20A, to a receiving unit 2060, see FIG. 20B. Assuch, an exemplary method of repelling the transfer media 2019 from thedistal opening of one or more channels 2015 in this instance comprises:applying a voltage potential between the one or more conducting sheets2024 and the structure 2005, via a power unit 2022. Further, in thiscase, the transfer media 2019 may contain a positive or negative chargewhich reacts to an electrostatic or magnetic field or a potentialdifference created by the power unit 2022, as it passes through thestructure 2005, and a sheet 2024. Additionally, the electrostaticproperties of the one or more conducting sheets 2024 and the structure2005 can be tuned to optimize the transfer of the polynucleotide 2017 inthe transfer media 2019 through a channel 2015 in the structure.Finally, a nonconductive separator may be positioned between thestructure 2005 and a conducting sheet 2024, to tune or optimize theelectrostatic or magnetic field or the potential difference formedtherein.

In some arrangements, a combination of means for attracting the transfermedia to or from the channels employ a fluid or gas transfer mechanismsincluding but not limited to: to laminar pressure, capillary pressure,slip flow pressure, magnetic force, electrostatic force, peristalticforce, sound waves, vibrational force, centripetal force, centrifugalforce, or any combination thereof.

In some instance, see e.g., FIG. 21 the receiving unit 2160 comprisestwo or more compartments 2162 a 2162 b, wherein each compartment 2162 a2162 b is capable of receiving and temporarily storing a singlerespective portion of a gaseous or fluidic transfer media 2119comprising a polynucleotide 2117. In other arrangements, see e.g., FIG.22 the receiving unit 2260 comprises a single compartment 2262, capableof receiving and temporarily storing one or more portions of a gaseousor fluidic transfer media 2219, comprising a polynucleotide 2217.

Sequencing

After extraction and/or amplification of polynucleotides from thesurface of the structure, suitable sequencing technology may be employedto sequence the polynucleotides. In some cases, the DNA sequence is readon the substrate or within a feature of a structure. In some cases, thepolynucleotides stored on the substrate are extracted is optionallyassembled into longer nucleic acids and then sequenced.

Polynucleotides synthesized and stored on the structures describedherein encode data that can be interpreted by reading the sequence ofthe synthesized polynucleotides and converting the sequence into binarycode readable by a computer. In some cases the sequences requireassembly, and the assembly step may need to be at the nucleic acidsequence stage or at the digital sequence stage.

Provided herein are detection systems comprising a device capable ofsequencing stored polynucleotides, either directly on the structureand/or after removal from the main structure. In cases where thestructure is a reel-to-reel tape of flexible material, the detectionsystem comprises a device for holding and advancing the structurethrough a detection location and a detector disposed proximate thedetection location for detecting a signal originated from a section ofthe tape when the section is at the detection location. In someinstances, the signal is indicative of a presence of a polynucleotide.In some embodiments, the signal is indicative of a sequence of apolynucleotide (e.g., a fluorescent signal). In some instances,information encoded within polynucleotides on a continuous tape is readby a computer as the tape is conveyed continuously through a detectoroperably connected to the computer. In some instances, a detectionsystem comprises a computer system comprising a polynucleotidesequencing device, a database for storage and retrieval of data relatingto polynucleotide sequence, software for converting DNA code of apolynucleotide sequence to binary code, a computer for reading thebinary code, or any combination thereof. Computer Systems

In various aspects, any of the systems described herein are operablylinked to a computer and are optionally automated through a computereither locally or remotely. In various embodiments, the methods andsystems of the invention further comprise software programs on computersystems and use thereof. Accordingly, computerized control for thesynchronization of the dispense/vacuum/refill functions such asorchestrating and synchronizing the material deposition device movement,dispense action and vacuum actuation are within the bounds of theinvention. In some instances, the computer systems are programmed tointerface between the user specified base sequence and the position of amaterial deposition device to deliver the correct reagents to specifiedregions of the substrate.

The computer system 2300 illustrated in FIG. 23 may be understood as alogical apparatus that can read instructions from media 2311 and/or anetwork port 2305, which can optionally be connected to server 2309having fixed medial 412. The system, such as shown in FIG. 4 can includea CPU 2301, disk drives 2303, optional input devices such as keyboard2315 and/or mouse 2316 and optional monitor 2307. Data communication canbe achieved through the indicated communication medium to a server at alocal or a remote location. The communication medium can include anymeans of transmitting and/or receiving data. For example, thecommunication medium can be a network connection, a wireless connectionor an internet connection. Such a connection can provide forcommunication over the World Wide Web. It is envisioned that datarelating to the present disclosure can be transmitted over such networksor connections for reception and/or review by a party 2322.

FIG. 24 is a block diagram illustrating a first example architecture ofa computer system 1500 that can be used in connection with exampleembodiments of the present invention.

As depicted in FIG. 5, the example computer system can include aprocessor 2402 for processing instructions. Non-limiting examples ofprocessors include: Intel Xeon™ processor, AMD Opteron™ processor.Samsung 32-bit RISC ARM 1176JZ(F)-S v1.0™ processor, ARM Cortex-A8Samsung SSPC100™ processor, ARM Cortex-A8 Apple A4™ processor, MarvellPXA 930™ processor, or a functionally-equivalent processor. Multiplethreads of execution can be used for parallel processing. In someembodiments, multiple processors or processors with multiple cores canalso be used, whether in a single computer system, in a cluster, ordistributed across systems over a network comprising a plurality ofcomputers, cell phones, and/or personal data assistant devices.

As illustrated in FIG. 24, a high speed cache 2404 can be connected to,or incorporated in, the processor 2402 to provide a high speed memoryfor instructions or data that have been recently, or are frequently,used by processor 2402. The processor 502 is connected to a north bridge2406 by a processor bus 2408. The north bridge 506 is connected torandom access memory (RAM) 2410 by a memory bus 2412 and manages accessto the RAM 2410 by the processor 2402. The north bridge 2406 is alsoconnected to a south bridge 2414 by a chipset bus 2416. The south bridge2414 is, in turn, connected to a peripheral bus 2418. The peripheral buscan be, for example, PCI, PCI-X, PCI Express, or other peripheral bus.The north bridge and south bridge are often referred to as a processorchipset and manage data transfer between the processor, RAM, andperipheral components on the peripheral bus 2418. In some alternativearchitectures, the functionality of the north bridge can be incorporatedinto the processor instead of using a separate north bridge chip.

In some embodiments, system 2400 can include an accelerator card 2422attached to the peripheral bus 2418. The accelerator can include fieldprogrammable gate arrays (FPGAs) or other hardware for acceleratingcertain processing. For example, an accelerator can be used for adaptivedata restructuring or to evaluate algebraic expressions used in extendedset processing.

Software and data are stored in external storage 2424 and can be loadedinto RAM 2410 and/or cache 2404 for use by the processor. The system2400 includes an operating system for managing system resources;non-limiting examples of operating systems include: Linux, Windows™,MACOS™, BlackBerry OS™, iOS™, and other functionally-equivalentoperating systems, as well as application software running on top of theoperating system for managing data storage and optimization inaccordance with example embodiments of the present invention.

In this example, system 2400 also includes network interface cards(NICs) 2420 and 521 connected to the peripheral bus for providingnetwork interfaces to external storage, such as Network Attached Storage(NAS) and other computer systems that can be used for distributedparallel processing.

FIG. 25 is a diagram showing a network 2500 with a plurality of computersystems 602 a, and 602 b, a plurality of cell phones and personal dataassistants 2002 c, and Network Attached Storage (NAS) 2504 a, and 2504b. In example embodiments, systems 2502 a, 2502 b, and 2502 c can managedata storage and optimize data access for data stored in NetworkAttached Storage (NAS) 2504 a and 2504 b. A mathematical model can beused for the data and be evaluated using distributed parallel processingacross computer systems 2502 a, and 2502 b, and cell phone and personaldata assistant systems 2502 c. Computer systems 2502 a, and 2502 b, andcell phone and personal data assistant systems 2502 c can also provideparallel processing for adaptive data restructuring of the data storedin Network Attached Storage (NAS) 2504 a and 2504 b. FIG. 25 illustratesan example only, and a wide variety of other computer architectures andsystems can be used in conjunction with the various embodiments of thepresent invention. For example, a blade server can be used to provideparallel processing. Processor blades can be connected through a backplane to provide parallel processing. Storage can also be connected tothe back plane or as Network Attached Storage (NAS) through a separatenetwork interface.

In some example embodiments, processors can maintain separate memoryspaces and transmit data through network interfaces, back plane or otherconnectors for parallel processing by other processors. In otherembodiments, some or all of the processors can use a shared virtualaddress memory space.

FIG. 26 is a block diagram of a multiprocessor computer system 2600using a shared virtual address memory space in accordance with anexample embodiment. The system includes a plurality of processors 2602a-f that can access a shared memory subsystem 2604. The systemincorporates a plurality of programmable hardware memory algorithmprocessors (MAPs) 2606 a-f in the memory subsystem 2604. Each MAP 2606a-f can comprise a memory 2608 a-f and one or more field programmablegate arrays (FPGAs) 2610 a-f. The MAP provides a configurable functionalunit and particular algorithms or portions of algorithms can be providedto the FPGAs 2610 a-f for processing in close coordination with arespective processor. For example, the MAPs can be used to evaluatealgebraic expressions regarding the data model and to perform adaptivedata restructuring in example embodiments. In this example, each MAP isglobally accessible by all of the processors for these purposes. In oneconfiguration, each MAP can use Direct Memory Access (DMA) to access anassociated memory 2608 a-f, allowing it to execute tasks independentlyof, and asynchronously from, the respective microprocessor 2602 a-f Inthis configuration, a MAP can feed results directly to another MAP forpipelining and parallel execution of algorithms.

The above computer architectures and systems are examples only, and awide variety of other computer, cell phone, and personal data assistantarchitectures and systems can be used in connection with exampleembodiments, including systems using any combination of generalprocessors, co-processors, FPGAs and other programmable logic devices,system on chips (SOCs), application specific integrated circuits(ASICs), and other processing and logic elements. In some embodiments,all or part of the computer system can be implemented in software orhardware. Any variety of data storage media can be used in connectionwith example embodiments, including random access memory, hard drives,flash memory, tape drives, disk arrays, Network Attached Storage (NAS)and other local or distributed data storage devices and systems.

In example embodiments, the computer system can be implemented usingsoftware modules executing on any of the above or other computerarchitectures and systems. In other embodiments, the functions of thesystem can be implemented partially or completely in firmware,programmable logic devices such as field programmable gate arrays(FPGAs) as referenced in FIG. 7, system on chips (SOCs), applicationspecific integrated circuits (ASICs), or other processing and logicelements. For example, the Set Processor and Optimizer can beimplemented with hardware acceleration through the use of a hardwareaccelerator card, such as the accelerator card 1822 illustrated in FIG.18.

Provided herein are methods for storing information, comprising:converting an item of information in the form of at least one digitalsequence to at least one nucleic acid sequence; providing a flexiblestructure having a surface; synthesizing a plurality of polynucleotideshaving predetermined sequences collectively encoding for the at leastone nucleic acid sequence, wherein the plurality of polynucleotidescomprises at least about 100,000 polynucleotides, and wherein theplurality of polynucleotides extends from the surface of the flexiblestructure; and storing the plurality of polynucleotides. Furtherprovided herein are methods wherein synthesizing comprises: depositingnucleosides on the surface at predetermined locations; and moving leasta portion of the flexible structure through a bath or emissions from aspray bar. Further provided herein are methods wherein the bath oremissions from a spray bar expose the surface of the structure to anoxidizing reagent or a deblocking reagent. Further provided herein aremethods wherein synthesizing further comprises capping the nucleosidesdeposited on the surface. Further provided herein are methods whereinthe nucleosides comprise a nucleoside phosphoramidite. Further providedherein are methods wherein the flexible structure comprises areel-to-reel tape or a continuous tape. Further provided herein aremethods wherein the flexible structure comprises a thermoplasticmaterial. Further provided herein are methods wherein the thermoplasticmaterial comprises a polyaryletherketone. Further provided herein aremethods wherein the polyaryletherketone is polyetherketone,polyetherketoneketone, poly(ether ether ketone ketone), polyether etherketone or polyetherketoneetherketoneketone. Further provided herein aremethods wherein the flexible structure comprises nylon, nitrocellulose,polypropylene, polycarbonate, polyethylene, polyurethane, polystyrene,acetal, acrylic, acrylonitrile, butadiene styrene, polyethyleneterephthalate, polymethyl methacrylate, polyvinyl chloride, transparentPVC foil, Poly(methyl methacrylate), styrenic polymer,fluorine-containing polymers, polyethersulfone or polyimide. Furtherprovided herein are methods wherein each polynucleotide of the pluralityof polynucleotides comprises from 50 to 500 bases in length. Furtherprovided herein are methods wherein the plurality of polynucleotidescomprises at least about 10 billion polynucleotides. Further providedherein are methods wherein at least about 1.75×10¹³ nucleobases aresynthesized within 24 hours. Further provided herein are methods whereinat least about 262.5×10⁹ polynucleotides are synthesized within 72hours. Further provided herein are methods wherein the item ofinformation is text information, audio information or visualinformation. Further provided herein are methods wherein the nucleosidescomprise nucleoside phosphoramidite.

Provided herein are methods for storing information, comprising:converting an item of information in the form of at least one digitalsequence to at least one nucleic acid sequence; providing a structurehaving a surface; synthesizing a plurality of polynucleotides havingpredetermined sequences collectively encoding for the at least onenucleic acid sequence, wherein the plurality of polynucleotidescomprises at least about 100,000 polynucleotides, wherein the pluralityof polynucleotides extends from the surface of the structure, andwherein synthesizing comprises: cleaning a surface of the structure;depositing nucleosides on the surface at predetermined locations;oxidizing, deblocking, and optionally capping the nucleosides depositedon the surface; wherein the cleaning, oxidizing, deblocking, and cappingcomprises moving at least a portion of the flexible structure through abath or emissions from a spray bar: and storing the plurality ofpolynucleotides. Further provided herein are methods wherein thenucleosides comprise nucleoside phosphoramidite.

Provided herein are methods for storing information, comprising:converting an item of information in the form of at least one digitalsequence to at least one nucleic acid sequence; synthesizing a pluralityof polynucleotides having predetermined sequences collectively encodingfor the at least one nucleic acid sequence, wherein the plurality ofpolynucleotides comprises at least about 10,000 polynucleotides, whereinthe plurality of polynucleotides collectively encode for a sequence thatdiffers from the predetermined sequences by no more than 1 base in 1000,and wherein each polynucleotide of the plurality of polynucleotidescomprises from 50 to 500 bases in length; and storing the at least about10,000 polynucleotides. Further provided herein are methods wherein theplurality of polynucleotides comprises at least about 100,000polynucleotides. Further provided herein are methods wherein theplurality of polynucleotides comprises at least about 1,000,000polynucleotides. Further provided herein are methods wherein theplurality of polynucleotides comprises at least about 10 billionpolynucleotides. Further provided herein are methods wherein greaterthan 90% of the polynucleotides encode for a sequence that does notdiffer from the predetermined sequence. Further provided herein aremethods wherein the item of information is text information, audioinformation or visual information. Further provided herein are methodswherein the structure is rigid or flexible, and wherein the structurecomprises a surface, and wherein the plurality of polynucleotides extendfrom the surface. Further provided herein are methods wherein thenucleosides comprise nucleoside phosphoramidite.

Provided herein are methods for storing information, comprising:converting an item of information in the form of at least one digitalsequence to at least one nucleic acid sequence; synthesizing a pluralityof polynucleotides having predetermined sequences collectively encodingfor the at least one nucleic acid sequence, wherein the plurality ofpolynucleotides comprises at least about 10,000 polynucleotides, whereineach polynucleotide of the plurality of polynucleotides comprises from50 to 500 bases in length, and where the plurality of polynucleotidesextends from the surface of a flexible structure, and storing theplurality of polynucleotides. Further provided herein are methodswherein the flexible structure comprises a reel-to-reel tape or acontinuous tape. Further provided herein are methods wherein eachpolynucleotide extends from a locus on the surface of the flexiblestructure, wherein the locus is about 1 um to about 500 um in diameter.Further provided herein are methods wherein the locus is about 1 um toabout 50 um in diameter. Further provided herein are methods wherein thelocus is about 10 um in diameter. Further provided herein are methodswherein the flexible structure comprises a thermoplastic material.Further provided herein are methods wherein the thermoplastic materialcomprises a polyaryletherketone. Further provided herein are methodswherein the polyaryletherketone is polyetherketone,polyetherketoneketone, poly(ether ether ketone ketone), polyether etherketone or polyetherketoneetherketoneketone. Further provided herein aremethods wherein the flexible structure comprises nylon, nitrocellulose,polypropylene, polycarbonate, polyethylene, polyurethane, polystyrene,acetal, acrylic, acrylonitrile, butadiene styrene, polyethyleneterephthalate, polymethyl methacrylate, polyvinyl chloride, transparentPVC foil, Poly(methyl methacrylate), styrenic polymer,fluorine-containing polymers, polyethersulfone or polyimide. Furtherprovided herein are methods wherein the flexible structure has athickness of less than about 10 mm. Further provided herein are methodswherein each polynucleotide is about 200 bases in length. Furtherprovided herein are methods wherein at least about 1.75×10¹³ nucleobasesare synthesized within 24 hours. Further provided herein are methodswherein at least about 262.5×10⁹ polynucleotides are synthesized within72 hours. Further provided herein are methods wherein the nucleosidescomprise nucleoside phosphoramidite.

Provided herein are methods for storing information, the methodcomprising, encrypting at least one item of information in the form ofat least one digital sequence to at least one nucleic acid sequence;synthesizing a plurality of polynucleotides having predeterminedsequences collectively encoding for the at least one nucleic acidsequence, wherein the plurality of polynucleotides comprises at leastabout 10,000 polynucleotides, and wherein each polynucleotide of theplurality of polynucleotides comprises from 50 to 500 bases in length;storing the plurality of polynucleotides; sequencing the plurality ofpolynucleotides; decrypting the plurality of polynucleotides from anucleic acid sequence to a digital sequence; and assembling the digitalsequence to form the at least one digital sequence, wherein the at leastone digital sequence is assembled with 100% accuracy compared to theinitial at least one digital sequence. Further provided herein aremethods further comprising releasing the plurality of polynucleotides.Further provided herein are methods wherein the nucleosides comprisenucleoside phosphoramidite.

Provided herein are devices for information storage, comprising: aflexible structure having a surface; and a plurality of loci on thesurface, wherein each locus has a width of from about 1 to about 500 um,and wherein each locus of the plurality of loci is coated with a moietythat binds to the surface and comprises a hydroxyl group available fornucleoside coupling. Further provided herein are devices wherein theflexible structure rests in a curved position. Further provided hereinare devices wherein the curved position comprises a curve that isgreater than 30 degrees. Further provided herein are devices wherein thecurved position comprises a curve that is greater than 180 degrees.Further provided herein are devices wherein the flexible structurecomprises at least about 1 million loci. Further provided herein aredevices wherein the flexible structure has a total surface area of lessthan about 4.5 m². Further provided herein are devices wherein theflexible structure comprises more than 2 billion loci per m². Furtherprovided herein are devices wherein the flexible structure comprises athermoplastic material. Further provided herein are devices wherein thethermoplastic material comprises a polyaryletherketone. Further providedherein are devices wherein the polyaryletherketone is polyetherketone,polyetherketoneketone, poly(ether ether ketone ketone), polyether etherketone or polyetherketoneetherketoneketone. Further provided herein aredevices wherein the flexible structure comprises nylon, nitrocellulose,polypropylene, polycarbonate, polyethylene, polyurethane, polystyrene,acetal, acrylic, acrylonitrile, butadiene styrene, polyethyleneterephthalate, polymethyl methacrylate, polyvinyl chloride, transparentPVC foil, Poly(methyl methacrylate), styrenic polymer,fluorine-containing polymers, polyethersulfone or polyimide. Furtherprovided herein are devices wherein the flexible structure has athickness of less than about 10 mm. Further provided herein are deviceswherein each locus is from about 1 um to about 50 um in width. Furtherprovided herein are devices wherein each locus has a diameter of about10 um. Further provided herein are devices wherein the center of a firstlocus is about 21 um from the center of a second locus and the firstlocus and the second locus. Further provided herein are devices whereinthe flexible structure comprises a reel-to-reel tape or a continuoustape. Further provided herein are devices wherein each locus comprises achannel.

Provided herein are polynucleotide libraries for information storage,comprising a plurality of polynucleotides, wherein the plurality ofpolynucleotides comprises at least about 10,000 polynucleotides, whereinthe plurality of polynucleotides collectively encodes for a sequencethat differs from an aggregate of predetermined sequences by no morethan 1 base in 1000, and wherein each polynucleotide of the plurality ofpolynucleotides comprises: a predetermined sequence that, whendecrypted, encodes for digital information. Further provided herein arelibraries wherein the plurality of polynucleotides comprises at leastabout 100,000 polynucleotides. Further provided herein are librarieswherein the plurality of polynucleotides comprises at least about 10billion polynucleotides. Further provided herein are libraries whereineach polynucleotide of the plurality of polynucleotides is attached to asurface of a structure by a tether. Further provided herein arelibraries wherein the tether comprises a cleavable region having atleast one nucleotide chemically modified to detach from thepolynucleotide in the presence of a cleaving reagent. Further providedherein are libraries wherein the tether comprises from about 10 to about50 bases. Further provided herein are libraries wherein greater than 90%of the polynucleotides encode for a sequence that does not differ fromthe predetermined sequences. Further provided herein are librarieswherein the digital information encodes for text, audio or visualinformation. Further provided herein are libraries wherein the libraryis synthesized in less than 3 days. Further provided herein arelibraries wherein the library is synthesized in less than 24 hours.

Further provided herein are methods for synthesizing polynucleotidesthat encode a range of an amount of digital information. In someinstances, an amount of digital information is at least 1 gigabyte (GB).In some instances, the amount of digital information is at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000 or more than 1000 gigabytes. In some instances, the amount ofdigital information is at least 1 terabyte (TB). In some instances, theamount of digital information is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more than1000 terabytes. In some instances, the amount of digital information isat least 1 petabyte (PB). In some instances, the amount of digitalinformation is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000 or more than 1000 petabytes.

The following examples are set forth to illustrate more clearly theprinciple and practice of embodiments disclosed herein to those skilledin the art and are not to be construed as limiting the scope of anyclaimed embodiments. Unless otherwise stated, all parts and percentagesare on a weight basis.

EXAMPLES Example 1: Functionalization of a Device Surface

A device was functionalized to support the attachment and synthesis of alibrary of polynucleotides. The device surface was first wet cleanedusing a piranha solution comprising 90% H₂SO₄ and 10% H₂O₂ for 20minutes. The device was rinsed in several beakers with DI water, heldunder a DI water gooseneck faucet for 5 min, and dried with N₂. Thedevice was subsequently soaked in NH₄OH (1:100; 3 mL:300 mL) for 5 min,rinsed with DI water using a handgun, soaked in three successive beakerswith DI water for 1 min each, and then rinsed again with DI water usingthe handgun. The device was then plasma cleaned by exposing the devicesurface to O₂. A SAMCO PC-300 instrument was used to plasma etch O₂ at250 watts for 1 min in downstream mode.

The cleaned device surface was actively functionalized with a solutioncomprising N-(3-triethoxysilylpropyl)-4-hydroxybutyramide using aYES-1224P vapor deposition oven system with the following parameters:0.5 to 1 torr, 60 min, 70° C., 135° C. vaporizer. The device surface wasresist coated using a Brewer Science 200X spin coater. SPR™ 3612photoresist was spin coated on the device at 2500 rpm for 40 sec. Thedevice was pre-baked for 30 min at 90° C. on a Brewer hot plate. Thedevice was subjected to photolithography using a Karl Suss MA6 maskaligner instrument. The device was exposed for 2.2 sec and developed for1 min in MSF 26A. Remaining developer was rinsed with the handgun andthe device soaked in water for 5 min. The device was baked for 30 min at100° C. in the oven, followed by visual inspection for lithographydefects using a Nikon L200. A cleaning process was used to removeresidual resist using the SAMCO PC-300 instrument to O₂ plasma etch at250 watts for 1 min.

The device surface was passively functionalized with a 100 μL solutionof perfluorooctyltrichlorosilane mixed with 10 μL light mineral oil. Thedevice was placed in a chamber, pumped for 10 min. and then the valvewas closed to the pump and left to stand for 10 min. The chamber wasvented to air. The device was resist stripped by performing two soaksfor 5 min in 500 ml, NMP at 70° C. with ultrasonication at maximum power(9 on Crest system). The device was then soaked for 5 min in 500 mLisopropanol at room temperature with ultrasonication at maximum power.The device was dipped in 300 mL of 200 proof ethanol and blown dry withN₂. The functionalized surface was activated to serve as a support forpolynucleotide synthesis.

Example 2: Synthesis of a 50-mer Sequence Oligonucleotides

A two dimensional oligonucleotide synthesis device was assembled into aflowcell, which was connected to a flowcell (Applied Biosystems (ABI394DNA Synthesizer”). The two-dimensional oligonucleotide synthesis devicewas uniformly functionalized withN-(3-TRIFTHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE (Gelest) was used tosynthesize an exemplary oligonucleotide of 50 bp (“50-meroligonucleotide”) using oligonucleotide synthesis methods describedherein.

The sequence of the 50-mer was as described in SEQ ID NO.: 1.5′AGACAATCAACCATITGGGGTGGACAGCCTTGACCrCTTAGACTTCGGCAT##TTTTT TTTTT3′(SEQ ID NO.: 1), where # denotes Thymidine-succinyl hexamide CEDphosphoramidite (CLP-2244 from ChemGenes), which is a cleavable linkerenabling the release of polynucleotides from the surface duringdeprotection.

The synthesis was done using standard DNA synthesis chemistry (coupling,capping, oxidation, and deblocking) according to the protocol in Table 3and an ABI synthesizer.

TABLE 3 General DNA Synthesis Process Name Process Step Time (sec) WASH(Acetonitrile Wash Acetonitrile System Flush 4 Flow) Acetonitrile toFlowcell 23 N2 System Flush 4 Acetonitrile System Flush 4 DNA BASEADDITION Activator Manifold Flush 2 (Phosphoramidite + Activator toFlowcell 6 Activator Flow) Activator + 6 Phosphoramidite to FlowcellActivator to Flowcell 0.5 Activator + 5 Phosphoramidite to FlowcellActivator to Flowcell 0.5 Activator + 5 Phosphoramidite to FlowcellActivator to Flowcell 0.5 Activator + 5 Phosphoramidite to FlowcellIncubate for 25 sec 25 WASH (Acetonitrile Wash Acetonitrile System Flush4 Flow) Acetonitrile to Flowcell 15 N2 System Flush 4 AcetonitrileSystem Flush 4 DNA BASE ADDITION Activator Manifold Flush 2(Phosphoramidite + Activator to Flowcell 5 Activator Flow) Activator +18 Phosphoramidite to Flowcell Incubate for 25 sec 25 WASH (AcetonitrileWash Acetonitrile System Flush 4 Flow) Acetonitrile to Flowcell 15 N2System Flush 4 Acetonitrile System Flush 4 CAPPING (CapA + B, 1:1,CapA + B to Flowcell 15 Flow) WASH (Acetonitrile Wash AcetonitrileSystem Flush 4 Flow) Acetonitrile to Flowcell 15 Acetonitrile SystemFlush 4 OXIDATION (Oxidizer Oxidizer to Flowcell 18 Flow) WASH(Acetonitrile Wash Acetonitrile System Flush 4 Flow) N2 System Flush 4Acetonitrile System Flush 4 Acetonitrile to Floweell 15 AcetonitrileSystem Flush 4 Acetonitrile to Flowcell 15 N2 System Flush 4Acetonitrile System Flush 4 Acetonitrile to Flowcell 23 N2 System Flush4 Acetonitrile System Flush 4 DEBLOCKING (Deblock Deblock to Flowcell 36Flow) WASH (Acetonitrile Wash Acetonitrile System Flush 4 Flow) N2System Flush 4 Acetonitrile System Flush 4 Acetonitrile to Flowcell 18N2 System Flush 4.13 Acetonitrile System Flush 4.13 Acetonitrile toFlowcell 15

The phosphoramidite/activator combination was delivered similar to thedelivery of bulk reagents through the flowcell. No drying steps wereperformed as the environment stays “wet” with reagent the entire time.

The flow restrictor was removed from the ABI 394 synthesizer to enablefaster flow. Without flow restrictor, flow rates for amidites (0.1M inACN), Activator. (0.25M Benzoylthiotetrazole (“BTT”, 30-3070-xx fromGlenResearch) in ACN), and Ox (0.02M I2 in 20% pyridine, 10% water, and70% THF) were roughly ˜100 uL/sec, for acetonitrile (“ACN”) and cappingreagents (1:1 mix of CapA and CapB, wherein CapA is acetic anhydride inTHF/Pyridine and CapB is 16% 1-methylimidizole in THF), roughly ˜200uL/sec, and for Deblock (3% dichloroacetic acid in toluene), roughly˜300 uL/sec (compared to ˜50 ul/sec for all reagents with flowrestrictor). The time to completely push out Oxidizer was observed, thetiming for chemical flow times was adjusted accordingly and an extra ACNwash was introduced between different chemicals. After oligonucleotidesynthesis, the chip was deprotected in gaseous ammonia overnight at 75psi. Five drops of water were applied to the surface to assemblepolynucleotides. The assembled polynucleotides were then analyzed on aBioAnalyzer small RNA chip (data not shown).

Example 3: Synthesis of a 100-Mer Sequence Oligonucleotides

The same process as described in Example 2 for the synthesis of the50-mer sequence was used for the synthesis of a 100-mer oligonucleotide(“100-mer oligonucleotide”; 5′CGGGATCCTTATCGTCATCGTCGTACAGATCCCGACCCATTGCTGTCCACCAGTCATGCTAGCCATACCATGATGATGATGATGATGAGAACCCCGCAT##TTTTTTTTTT3′, where #denotes Thymidine-succinyl hexamide CED phosphoramidite (CLP-2244 fromChemGenes); SEQ ID NO.: 2) on two different silicon chips, the first oneuniformly functionalized withN-(3-TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE and the second onefunctionalized with 5/95 mix of 11-acetoxyundecyltriethoxysilane andn-decyltriethoxysilane, and the polynucleotides extracted from thesurface were analyzed on a BioAnalyzer instrument (data not shown).

All ten samples from the two chips were further PCR amplified using aforward (5′ATGCGGGTTCTCATCATC3′; SEQ ID NO.: 3) and a reverse(5′CGGGATCCTATCGTCATCG3′; SEQ ID NO.: 4) primer in a 50 uL PCR mix (25uL NEB QS mastermix, 2.5 uL 10 uM Forward primer, 2.5 uL 10 uM Reverseprimer, 1 uL polynucleotide extracted from the surface, and water up to50 uL) using the following thermalcycling program:

-   -   98 C, 30 sec    -   98 C, 10 sec; 63 C, 10 sec; 72 C, 10 sec; repeat 12 cycles    -   72 C, 2 min

The PCR products were also run on a BioAnalyzer (data not shown),demonstrating sharp peaks at the 100-mer position. Next, the PCRamplified samples were cloned, and Sanger sequenced. Table 4 summarizesthe results from the Sanger sequencing for samples taken from spots 1-5from chip 1 and for samples taken from spots 6-10 from chip 2.

TABLE 4 Spot Error rate Cycle efficiency 1 1/763 bp 99.87% 2 1/824 bp99.88% 3 1/780 bp 99.87% 4 1/429 bp 99.77% 5 1/1525 bp  99.93% 6 1/615bp 99.94% 7 1/531 bp 99.81% 8 1/1769 bp  99.94% 9 1/854 bp 99.88% 101/1451 bp  99.93%

Thus, the high quality and uniformity of the synthesizedoligonucleotides were repeated on two chips with different surfacechemistries. Overall, 89%, corresponding to 233 out of 262 of the100-mers that were sequenced were perfect sequences with no errors.

Table 5 summarizes error characteristics for the sequences obtained fromthe oligonucleotides samples from spots 1-10.

TABLE 5 Sample ID/Spot OSA_ OSA_ no. 0046/1 0047/2 OSA_0048/3 OSA_0049/4OSA_0050/5 OSA_0051/6 OSA_0052/7 OSA_0053/8 OSA_0054/9 OSA_055/10 Total 32  32 32  32  32  32  32  32  32  32  Sequences Sequencing 25 of 27 of26 of 21 of 25 of 29 of 27 of 29 of 28 of 25 of 28 Quality 25 27 30 2326 30 31 31 29 Oligo 23 of 25 of 22 of 18 of 24 of 25 of 22 of 28 of 26of 20 of 25 Quality 25 27 26 21 25 29 27 29 28 ROI 2500 2698 2561  2122   2499   2666   2625   2899   2798   2348   Match Count ROI   2   21 3 1 0 2 1 2 1 Mutation ROI Multi   0   0 0 0 0 0 0 0 0 0 Base DeletionROI Small   1   0 0 0 0 0 0 0 0 0 Insertion ROI   0   0 0 0 0 0 0 0 0 0Single Base Deletion Large   0   0 1 0 0 1 1 0 0 0 Deletion CountMutation:   2   2 1 2 1 0 2 1 2 1 G > A Mutation:   0   0 0 1 0 0 0 0 00 T > C ROI Error   3   2 2 3 1 1 3 1 2 1 Count ROI Error Err. ~1 Err.~1 Err. ~1 Err. ~1 Err. ~1 Err. ~1 Err. ~1 Err. ~1 Err. ~1 Err. ~1 Ratein 834 in 1350 in 1282 in 708 in 2500 in 2667 in 876 in 2900 in 1400 in2349 ROI MP MP MP MP MP MP MP MP MP MP Minus Err. ~1 Err. ~1 Err. ~1Err. ~1 Err. ~1 Err. ~1 Err. ~1 Err. ~1 Err. ~1 Err. ~1 Primer in 763 in824 in 780 in 429 in 1525 in 1615 in 531 in 1769 in 854 in 1451 ErrorRate

Example 4: Highly Accurate DNA-Based Information Storage and Assembly

Digital information was selected in the form of binary data totalingabout 0.2 GB included content for the Universal Declaration or HumanRights in more than 100 languages, the top 100) books of ProjectGuttenberg and a seed database. The digital, information was encryptedinto a nucleic acid-based sequence and divided into strings. Over 10million non-identical polynucleotides, each corresponding to a string,were synthesized on a rigid silicon surface in a manner similar to thatdescribed in Example 2. Each non-identical polynucleotide was underequal or less than 200 bases in length. The synthesized polynucleotideswere collected and sequenced and decoded back to digital code, with 100%accuracy for the source digital information, compared to the initial atleast one digital sequence.

Example 5: Conversion of Digital Information to Nucleic Acid Sequence

A computer txt file includes text information. A general purposecomputer uses a software program having machine instructions forconversion of the sequence to base 3, 4, or 5 sequence, depending oninstructions received. Each number in base 3 is assigned a nucleic acid(e.g., A=0, T=1, C=2). Each number in base 4 is assigned a nucleic acid(e.g., A=0, T=1, C=2. G=3). Alternatively, a base 5 quinary sequence isused, where each number in base 5 is assigned a nucleic acid (e.g., A=0,T=1, C=2, G=3, U=4). A sequence is generated as depicted in Table 6.Machine instructions are then provided for de novo synthesis ofpolynucleotides encoding the nucleic acid sequence.

TABLE 6 Text Jack went up the hill. Binary010010100110000101100011011010110010000001110111 sequence011001010110111001110100001000000111010101110000001000000111010001101000011001010010000001101000011010010110110001101100001011100000110100001010 0000110100001010Ternary 101010201100022101010021102012221200101112202210 sequence00212200221020001111221210201120102111212220010111000100200102200222222110022222112 Quaternary102212011203122302001313121112321310020013111300 sequence020013101220121102001220122112301230023200310022 00310022 Quinary332214330133012303013123001030244433343300431224 sequence10302032021020112342341100431241100334213

Example 6: Flexible Surface Having a High Density of Loci

A flexible structure comprising thermoplastic material is coated with anucleoside coupling reagent. The coating agent is patterned for a highdensity of loci. A portion of the flexible surface is illustrated inFIG. 11A. Each locus has a diameter of 10 um, with a center-to-centerdistance between two adjacent loci of 21 um. The locus size issufficient to accommodate a sessile drop volume of 0.2 pl during apolynucleotide synthesis deposition step. The small locus dimensionsallow for a high density of polynucleotides to be synthesized on thesurface of the substrate. The locus density is 2.2 billion loci/m² (1locus/441×10⁻¹² m²). A 4.5 m² substrate is manufactured having 10billion loci, each with a 10 um diameter. The flexible structure isoptionally placed in a continuous loop system. FIG. 9A, or areel-to-reel system. FIG. 9B, for polynucleotide synthesis.

Example 7: Polynucleotide Synthesis on a Flexible Structure

A flexible structure is prepared comprising a plurality of loci on athermoplastic flexible material. The structure serves as a support forthe synthesis of polynucleotides using a polynucleotide synthesis devicecomprising a deposition device. The flexible structure is in the form ofa flexible media much like a magnetic reel-to-reel tape.

De novo synthesis operates in a continuous production line manner withthe structure travelling through a solvent bath and then beneath a stackof printheads where the phosphoramidites are printed on to a surface ofthe structure. The flexible structure with the sessile drops depositedon to the surface is rolled into a bath of oxidizing agent, then thetape emerges from the oxidizing bath and is immersed in an acetonitrilewash bath then submerged in a deblock bath. Optionally, the tape istraversed through a capping bath. In an alternative workflow, theflexible structure emerges from the oxidizing bath and is sprayed withacetonitrile in a wash step.

Alternatively, a spray bar is used instead of a liquid bath. In thisprocess, the nucleotides are still deposited on the surface with aninkjet device but the flood steps are now done in a chamber with spraynozzles. For example, the deposition device has 2,048 nozzles that eachdeposit 100,000 droplets per second at 1 nucleobase per droplet. Thereis a sequential ordering of spray nozzles to mimic the ordering of theflood steps in standard phosphoramidite chemistry. This techniqueprovides for easily changing the chemicals loaded in the spray bar toaccommodate different process steps. Polynucleotides are deprotected orcleaved in the same manner as described in Example 2.

For each deposition device, more than 1.75×10¹³ nucleobases aredeposited on the structure per day. A plurality of 200 nucleobasepolynucleotides is synthesized. In 3 days, at a rate of 1.75×10¹³ basesper day, 262.5×10⁹ polynucleotides are synthesized. Each oligonucleotidesequence comprises a polynucleotide of at least 15 bases embedded in alonger polynucleotide. In one instance, the polynucleotide is designedto have at least, in 5′ to 3′ order, a linker region, cleavage region, afirst primer binding region, a bar code region, target sequence regions,and a second primer region.

Example 8: Electrostatic Transfer of Polynucleotides Following De NovoSynthesis

Polynucleotides are synthesized similarly to Examples 2-3. Followingpolynucleotide synthesis, the polynucleotides are transferred from achannel in a structure to one or more channels or a receiving unit usingelectrostatic force.

An aqueous or gaseous transfer media that adheres to the polynucleotidesis deposited. The channel is surrounded by interconnected conductorplates located above the channel. The transfer media comprises a charge(positive or negative) that reacts with an electrostatic field createdby the conductor plates. A voltage potential is applied between theinterconnected conductor plates, resulting in attraction of the transfermedia and transfer of the polynucleotides through an opening of thechannel.

In order to repel the polynucleotides from the channel, the channel issurrounded by interconnected conductor plates located below the channel.When voltage potential is applied between the interconnected conductorplates, the transfer media is repelled from the channel to one or morechannels or the receiving unit.

Example 9: Transfer of Polynucleotides Following De Novo Synthesis UsingVibrational Force

Polynucleotides are synthesized similarly to Examples 2-3. Followingpolynucleotide synthesis, the polynucleotides are transferred from achannel in a structure to one or more channels or a receiving unit usingvibrational force.

An aqueous or gaseous transfer media that adheres to the polynucleotidesis deposited. The channel is surrounded by vibrational energyapplicators. Vibrational energy is applied through the vibrationalenergy applicators, resulting in transfer of the polynucleotides throughan opening of the channel to one or more channels or the receiving unit.

Example 10: Transfer of Polynucleotides Following De Novo SynthesisUsing a Slip

Polynucleotides are synthesized similarly to Examples 2-3. Followingpolynucleotide synthesis, the polynucleotides are transferred from achannel in a structure to one or more channels or a receiving unit usinga slip.

An aqueous or gaseous transfer media that adheres to the polynucleotidesis deposited. A slip is positioned in contact with the structure at anangle. By rotating the slip relative to the structure, for example ateither 10°, 20°, 30°, 40°, 50°, 60°, 70° or 80°, the transfer media istransferred to one or more channels or the receiving unit.

Example 11: Transfer of Polynucleotides Following De Novo SynthesisUsing Applied Pressure

Polynucleotides are synthesized similarly to Examples 2-3. Followingpolynucleotide synthesis, the polynucleotides are transferred from achannel in a structure to one or more channels or a receiving unit usingapplied pressure.

An aqueous or gaseous transfer media that adheres to the polynucleotidesis deposited. An applied pressure within a gas or fluid and a pressurerelease are employed to force the transfer media through a channel inthe structure. By creating a pressure differential, opening of apressure release forces the transfer media through the channel.

Example 12: Transfer of Polynucleotides Following De Novo SynthesisUsing Applied Pressure and a Nozzle

Polynucleotides are synthesized similarly to Examples 2-3. Followingpolynucleotide synthesis, the polynucleotides are transferred from achannel in a flexible structure to one or more channels or a receivingunit using applied pressure and a nozzle.

An aqueous or gaseous transfer media that adheres to the polynucleotidesis deposited. The flexible structure is moved using a roller such thatthe channel is aligned below a nozzle. The nozzle then applies pressuretowards a channel and forces the transfer media through the channel.

Example 13: Transfer of Polynucleotides Following De Novo SynthesisUsing a Pin

Polynucleotides are synthesized similarly to Examples 2-3. Followingpolynucleotide synthesis, the polynucleotides are transferred from achannel in a structure to one or more channels or a receiving unit usinga pin.

An aqueous or gaseous transfer media that adheres to the polynucleotidesis deposited. The pin contacts and attracts the transfer media and arelative vertical motion between the pin and the structure dislocatesthe transfer media from the structure to one or more channels or thereceiving unit.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A method for storing information, the methodcomprising: (a) converting at least one item of information in a form ofat least one digital sequence to at least one nucleic acid sequence; (b)synthesizing a plurality of polynucleotides having predeterminedsequences collectively encoding for the at least one nucleic acidsequence, wherein each polynucleotide comprises: (i) a plurality ofcoding regions, wherein each coding region is identical; and (ii) atleast one non-coding region, wherein the at least one non-coding regioncomprises a cleavage region; and (c) storing the plurality ofpolynucleotides.
 2. The method of claim 1, wherein the cleavage regioncomprises a restriction enzyme recognition site.
 3. The method of claim1, wherein the cleavage region comprises a light sensitive nucleobase.4. The method of claim 1, further comprising application of arestriction enzyme, electromagnetic radiation, or a gaseous reagent tocleave at the cleavage region, thereby removing at least one of theplurality of coding regions.
 5. The method of claim 1, wherein eachcoding region comprises 25 to 500 bases in length.
 6. The method ofclaim 1, wherein each coding region comprises 10 to 2000 bases inlength.
 7. The method of claim 1, wherein each non-coding regioncomprises 1 to 10 bases in length.
 8. The method of claim 1, whereineach non-coding region comprises at most 200 bases.
 9. The method ofclaim 1, wherein the plurality of polynucleotides comprises at least10,000 polynucleotides.
 10. The method of claim 1, wherein the pluralityof polynucleotides comprises at least 1 billion polynucleotides.
 11. Themethod of claim 1, wherein greater than 90% of the polynucleotidesencode for a sequence that does not differ from the predeterminedsequence.
 12. The method of claim 1, wherein the at least one item ofinformation is text information, audio information or visualinformation.
 13. The method of claim 1, wherein a first non-codingregion within each polynucleotide has a different sequence than a secondnon-coding region within each polynucleotide.
 14. The method of claim 1,wherein each non-coding region within each polynucleotide has adifferent sequence.
 15. The method of claim 1, wherein a first cleavageregion within each polynucleotide has a different sequence than a secondcleavage region within each polynucleotide.
 16. The method of claim 1,wherein each cleavage region within each polynucleotide has a differentsequence.
 17. The method of claim 1, wherein a number of cleavageregions within each polynucleotide is at least 1, 2, 3, 4, or
 5. 18. Themethod of claim 17, wherein a sequence for the number of cleavageregions is different.
 19. The method of claim 1, wherein eachpolynucleotide comprises a tether region
 20. The method of claim 1,further comprising selectively transferring the plurality ofpolynucleotides to a receiving unit, wherein selectively transferringcomprises application of a force, wherein the force is laminar pressure,capillary pressure, slip flow pressure, magnetic force, electrostaticforce, peristaltic force, sound waves, vibrational force, centripetalforce, centrifugal force, or any combination thereof.